KR20230028496A - truffle-derived sweet protein - Google Patents

Abstract

A newly identified fungal sweet modified protein and cDNA encoding the protein are described. Specifically, the Myd protein active in activating sweetness and the cDNA encoding it are described along with methods for isolating this cDNA and isolating and expressing this protein. Also disclosed are uses of sweetening compositions comprising the proteins of the present invention and methods of providing improved flavor to products for oral administration.

Description

truffle-derived sweet protein

CROSS REFERENCES TO RELATED APPLICATIONS

This patent application claims priority to US Provisional Patent Application No. 63/044,245, filed on June 25, 2020, which is incorporated by reference.

Integration by reference of electronically submitted data

A list of computer readable nucleotide/amino acid sequences filed concurrently with this specification and identified as follows is incorporated herein by reference in its entirety: 86,678 byte ASCII named "338820_640_30A_WO_ST25.TXT" created on June 25, 2021 ( text) 1 file.

Excessive consumption of nutritive sweeteners has long been associated with diet-related health problems such as obesity, heart disease, metabolic disorders and dental problems. Therefore, consumers are increasingly looking for ways to reduce the amount of nutritive sweeteners in their diet. Manufacturers are responding to this demand by developing alternatives to nutritive sweeteners that can better mimic the desirable taste and functional properties of nutritive sweeteners.

Zero or low calorie sweeteners, preferably derived from natural sources, are required to limit the negative effects of high sugar consumption (eg diabetes and obesity). Commonly known zero or low calorie sweeteners include aspartame, acesulfame potassium, luo han guo (monk) fruit extract, neotame, saccharin, stevia and sucralose. However, these sweeteners have taste deficiencies such as bitterness.

Truffles are underground fruiting bodies of some ascomycetes, including genera belonging to the class Pezizales in the order Pezizomycetes. Truffles are ectomycorrhizal fungi, so they are usually found in close association with tree roots.

So far, seven sweets and flavors: brazzein, thaumatin, monellin, curculin, mabinlin, miraculin and pentadin. A protein that regulates is known. The key residues on the surface of proteins responsible for their biological activity have not yet been conclusively identified for these proteins. Monelin is 100,000 times sweeter than sucrose on a molar basis, followed by Brazzein and Thomasin, which are 500 and 3,000 times sweeter than sucrose on a gram basis. All of these proteins have been isolated from plants that grow in tropical rain forests. Most of them share no sequence homology or structural similarity, but thaumatin shares broad similarities at the protein sequence level with certain unsweetened proteins found in other plants. Proteins that regulate sweet taste are unknown in fungi.

There remains a need in the art to produce new low- or zero-calorie sweeteners with improved taste from natural sources. There is a need in the art to economically produce such sweetening compositions from potential sources of sweetening compositions, particularly Ascomycete fungal species.

Summary of Invention

The present invention relates to a newly identified fungal-derived sweet protein and the gene and cDNA encoding said protein, also referred to herein as MYD/Myd. More specifically, the present invention relates to newly identified sweet proteins, genes and cDNAs encoding the proteins, and methods of using these proteins, genes and cDNAs to adjust the taste of foods. The present invention provides, inter alia, a novel sweet protein identified herein as MYD1 and a DNA sequence encoding the corresponding polypeptide Myd1 (also referred to as mycodulcein). Myd1 is the first sweet protein identified in fungi. Myd1 reduces the sour, bitter or astringent taste of food and beverages, and additionally Myd1 has an activity to improve the taste of food and beverages, that is, to adjust the taste.

The present invention provides a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweetness modulating activity, wherein the polynucleotide sequence is (a) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75; (b) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:11, SEQ ID NO:11 by deletion, insertion, substitution or addition of up to 24 amino acids. a group consisting of a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 Encodes a polypeptide selected from

In one embodiment, the polypeptide sequence of a polypeptide having sweet taste regulating activity is the polypeptide sequence set forth in SEQ ID NO:3, or a polypeptide sequence having at least 80% sequence identity with SEQ ID NO:3. In another embodiment, the polypeptide encoding a polypeptide having sweet taste regulating activity is not a polypeptide of SEQ ID NO:3.

In another embodiment, the polypeptide sequence comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3.

The present invention also relates to (a) a polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:2 having at least one substitutional modification; (b) a polynucleotide comprising a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide is not a polynucleotide of SEQ ID NO:2, and (c) (i ) a polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 2 and (ii) a nucleotide sequence encoding a histidine tag, wherein the polynucleotide encodes a polypeptide having a sweetness regulating activity; a polynucleotide selected from the group consisting of Nucleotides (eg, isolated polynucleotides) are provided.

In one embodiment, the polypeptide sequence of a polypeptide having sweetness modulating activity comprises SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In particular, the polypeptide sequences of polypeptides having sweetness regulating activity are SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

In certain embodiments, the polynucleotide is SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67.

A polynucleotide encoding a polypeptide having sweetness regulatory activity is optionally operably linked to a heterologous regulatory element. Additionally or alternatively, the polynucleotide sequence further encodes a protein tag or label. The protein tag is optionally an affinity tag. The protein is optionally histidine tagged.

In one embodiment, the polynucleotide comprises SEQ ID NO: 20 corresponding to the coding sequence for His-tagged mycodulcein in E. coli, wherein residues 364-381 correspond to an optional His-tag sequence. SEQ ID NO: 20 is codon-optimized for expression in E. coli. In another embodiment, the polynucleotide comprises SEQ ID NO: 22 corresponding to the coding sequence for His-tagged mycodulcein in S. cerevisiae, wherein residues 364-381 correspond to any His-tagged sequence. . SEQ ID NO: 22 is codon optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO: 21 corresponds to His-tagged mycodulcein protein, wherein residues 122-127 correspond to any His tag sequence, and this polypeptide sequence is found in E. coli and S. cerevisiae. The same is true for the expression of

Expression cassettes comprising polynucleotides and vectors comprising the polynucleotides as well as host cells transformed with the vectors are provided. A method for producing a protein having a sweetness regulating activity is provided, comprising culturing a host cell in a medium under conditions for producing a protein having a sweetness regulating activity.

SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 A polypeptide comprising a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 (e.g., an isolated polypeptide peptide), wherein the polypeptide is optionally SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is optionally a protein. It further comprises a tag, in particular a histidine tag, wherein the polypeptide has sweetness regulating activity.

In one embodiment, the polypeptide comprises SEQ ID NO:3 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:3. In another aspect, the polypeptide is not a polypeptide of SEQ ID NO:3.

In another aspect, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3.

In one embodiment, the polypeptide (eg, the isolated polypeptide) is a polypeptide selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75 sequence, wherein the polypeptide is optionally not SEQ ID NO:3. In particular, the polypeptide is SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

The present invention provides a combination of (a) a product for oral administration, wherein the product is not a Matirolomyces terfezioides truffle, and (b) a sweetening composition comprising a polypeptide, wherein the The combination provides a composition comprising an enhanced sweet taste compared to products for oral administration. In one embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ and an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, said polypeptide optionally having SEQ ID NO:17 :3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 , SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide optionally is not a polypeptide of SEQ ID NO:3, wherein the polypeptide optionally It further comprises a histidine tag, and the polypeptide has sweetness regulating activity. For example, the polypeptide in the sweetener composition comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In particular, the polypeptide is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

In one embodiment, the present invention provides SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, A polypeptide comprising an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 It provides a sweetening composition comprising. wherein the polypeptide is optionally a polypeptide other than a polypeptide of the amino acid sequence of SEQ ID NO:3.

The present invention provides a method for adjusting the taste of an oral product comprising combining the product for oral administration with a sweetening composition comprising an effective amount of a polypeptide, wherein the product for oral administration is Matyrolomyces terpegioi It is not a death truffle, and the combination has an enhanced sweet taste compared to products for oral administration. In one embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ and an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; wherein the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide optionally comprises at least one substitution modification to a polypeptide sequence selected from the group consisting of SEQ ID NO:3 no, the polypeptide optionally further comprises a histidine tag, and the polypeptide has sweetness regulating activity. For example, the polypeptide in the sweetener composition comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 and optionally SEQ ID NO:3 does not consist of In particular, the polypeptide is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

Products for oral administration may be food, beverage, dietary supplement compositions or pharmaceutical compositions. Examples of foods include confectionery; sweet bakery products, ready-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bar; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spread; confectionery products; and sweetened breakfast cereals, but are not limited thereto. Examples of beverage products include carbonated beverages; non-carbonated beverages; and beverage concentrates, but are not limited thereto.

The present invention also relates to sweet taste control activity comprising the steps of (a) obtaining a composition comprising the polypeptide, and (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). It provides a method for purifying a polypeptide having. In one embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ and an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; The polypeptide is optionally SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide optionally comprises at least one substitution modification to a polypeptide sequence of SEQ ID NO:3 No, the polypeptide optionally further comprises a histidine tag, and the polypeptide has a sweetness regulating activity. For example, the polypeptide comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, optionally consisting of SEQ ID NO:3 It doesn't work. In particular, the polypeptide is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66 or SEQ ID NO: 68.

Other aspects and embodiments of the present invention will become apparent upon review of the drawings, detailed description and non-limiting examples herein.

Accordingly, the present invention provides isolated nucleic acid molecules encoding proteins capable of modulating the taste of sweetness and the polypeptides they encode. As described herein, the polypeptides of the present invention, alone or in combination with foods, beverages, dietary supplements or pharmaceuticals, mediate sweet taste perception. The present invention also provides compositions of isolated polypeptides capable of modifying a sweet taste, and the resulting combination having a sweet taste with a food, beverage, dietary supplement, or pharmaceutical composition. The present invention also provides methods for modifying the sweet taste of foods, beverages, dietary supplements or pharmaceutical compositions using the isolated polynucleotides and polypeptides of the present invention.

In one aspect of the present invention, a newly identified fungal sweet protein, herein designated Myd1, is provided. The term "Myd polypeptide" is used herein to identify any polypeptide according to the present invention that has at least 80% sequence identity to, for example, SEQ ID NO:3 and also has sweet-modifying activity. Myd polypeptides also include peptides SEQ ID NO:8 to SEQ ID NO:17 with sweetness modifying activity. A sweet-tasting, partially purified extract of M. terpegioides gleba was subjected to de novo amino acid sequencing to confirm the 20-mer N-terminal sequence (SEQ ID NO:4). The Myd1 coding sequence (presumptively derived from the MYD1 gene) was identified after the entire transcriptome of M. terpegioides gleba was de novo assembled using RNAseq reads. The entire transcriptome of M. terpegioides was screened using the 20-mer N-terminal sequence to identify transcripts predicted to encode proteins with 100% identity at the N-terminus. The identified transcript is predicted to encode a 121 amino acid protein. SEQ ID NO:1 was identified in this way. Start and stop codons were identified in the transcript to confirm the putative coding sequence SEQ ID NO:2. SEQ ID NO: 3 is a putative protein that is a 121 amino acid protein. The identity between the predicted protein SEQ ID NO:3 and the other protein sequences of GENBANK was less than 31%. The coding sequences for codon-optimized native mycodulcane for expression in E. coli and Saccharomyces cerevisiae correspond to the nucleic acid sequences of SEQ ID NO: 20 and SEQ ID NO: 22, respectively (any 6 residues amino acid sequence of SEQ ID NO: 3 with a histidine tag, ie encoding the amino acid sequence of SEQ ID NO: 21).

In one embodiment, a "Myd polypeptide" herein is at least 10% (e.g., 15%, 20%, 25%, 30%) as compared to a naturally occurring Myd polypeptide isolated from M. terpegioides gleba by extraction. %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of sweetness modification activity. have In another aspect, a "Myd polypeptide" herein has a sweetness modifying activity of at least 50% or more compared to a naturally occurring Myd polypeptide isolated from M. terpegioides gleba by extraction. In one embodiment, a “Myd polypeptide” herein has a sweet taste modification activity of at least 80% or greater compared to a naturally occurring Myd polypeptide isolated from M. terpegioides gleba by extraction. Sweetness modifying activity can be measured using any comparative method known in the art, in particular any method described in the Examples herein, more specifically using a sensory panel as described herein.

While not wishing to be bound by any particular theory, Myd1 is believed to be involved in sweet taste activation and is, for example, an agonist of taste 1 receptor 2 (T1R2) and/or taste 1 receptor 3 (T1R3). However, Myd1 can stimulate other taste receptors such as bitter, umami, sour and salty. The isolated or purified Myd polypeptide can be used in the food and pharmaceutical industries to customize taste, for example to adjust the sweetness of foods or drugs.

In a first aspect, the present invention includes a polynucleotide (e.g., an isolated polynucleotide) encoding a polypeptide having sweetness modulating activity, wherein the Singgi polypeptide sequence is (a) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ the amino acid sequence set forth in ID NO:17; (b) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85% %, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or more, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98% or at least 99%) amino acid sequences having sequence identity; and (c) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: No more than 24 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and any range thereof) Amino acid sequences modified by deletion, insertion, substitution or addition of amino acids It comprises, consists essentially of, or consists of a polypeptide selected from the group consisting of.

In some embodiments, the amino acid sequence of the polypeptide having sweetness modulating activity is an amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least 80% sequence identity to SEQ ID NO:3, or a deletion of 24 or fewer amino acids, An amino acid that has been modified in the amino acid sequence SEQ ID NO:3 by insertion, substitution or addition.

In certain embodiments, the present invention provides polynucleotides (e.g., isolated polynucleotides) encoding a polypeptide having sweetness modulating activity, wherein the polynucleotide sequence comprises (a) SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 a polypeptide sequence selected from the group; (b) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 from a polypeptide sequence that has been modified by deletion, insertion, substitution or addition of up to 24 amino acids from the group consisting of wherein the isolated polypeptide encoding a polypeptide having sweet taste regulatory activity is not a polypeptide of SEQ ID NO:3.

In another aspect, the invention comprises a polynucleotide (eg, an isolated polynucleotide), wherein the polynucleotide comprises (a) optionally at least one (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, SEQ ID NO: 2, including modifications (eg, deletions, insertions, substitutions or additions) of 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and ranges of such values A polynucleotide comprising the nucleic acid sequence described in; and (b) at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%) a nucleic acid sequence set forth in SEQ ID NO: 2 %, at least 89%, at least 90% or more, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity A polynucleotide selected from the group consisting of polynucleotides comprising a nucleic acid sequence having, optionally, the polynucleotide is not a polynucleotide of SEQ ID NO:2. In one embodiment, the polynucleotide encodes a polypeptide having sweetness modulating activity. In one embodiment, the amino acid sequence of the polypeptide having sweetness regulating activity is the amino acid sequence set forth in SEQ ID NO:3.

In one aspect, the invention provides a polynucleotide (eg, an isolated polynucleotide), wherein the polynucleotide sequence comprises (a) at least one (eg, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and ranges of these values) with substitution modifications nucleotide; (b) at least 90% (at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) a polynucleotide comprising a nucleic acid sequence having sequence identity, wherein the polynucleotide is optionally not a polynucleotide of SEQ ID NO:2, and (c) a nucleic acid sequence set forth in (i) SEQ ID NO:2 and (ii) a polynucleotide comprising a nucleotide sequence encoding a histidine tag, wherein the polynucleotide encodes a polypeptide having sweetness modulating activity.

The polynucleotides encoding Myd of the present invention may be in the form of single- or double-stranded DNA, RNA or artificial nucleic acids, or may be cDNA or chemically synthesized DNA that does not contain any introns.

The term “nucleic acid” or “nucleic acid sequence” refers to deoxy-ribonucleotides or ribonucleotide oligonucleotides in either single-stranded or double-stranded form. The term includes nucleic acids containing known analogues of natural nucleotides, ie oligonucleotides. The term also includes nucleic acid-like structures with synthetic backbones (e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992) Milligan J. Med. Chem. 96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); ) reference).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved, for example, by generating sequences in which the third position of one or more selected codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081). (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide and polynucleotide.

The present invention also provides an expression cassette comprising a polynucleotide encoding a Myd polypeptide and a host cell transformed with the vector.

In another aspect, the present invention relates to SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%) a polypeptide sequence selected from the group consisting of ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 %, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, (e.g., an isolated polypeptide) comprising, consisting essentially of, or consisting of a polypeptide sequence having at least 97%, at least 98% or at least 99% sequence identity, wherein the polypeptide is optionally at least one (eg, , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and a range of these values. , deletion, insertion, substitution or addition), wherein the polypeptide optionally further comprises a histidine tag, wherein the polypeptide has sweetness modulating activity.

The term "consisting essentially of" means a non-caking agent, filler, stabilizer (heat stabilizer, etc.), bulking agent (e.g., maltodextrose, acacia gum, etc.) that is not essential to, and does not significantly affect the function or activity of the product. It is allowed to contain ingredients that are not included.

The polypeptide comprises SEQ ID NO: 3 or at least 80% sequence identity with SEQ ID NO: 3. The polypeptide comprises SEQ ID NO: 8 or at least 80% sequence identity to SEQ ID NO: 8. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 9. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 10. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 11. The polypeptide comprises SEQ ID NO: 12 or at least 80% sequence identity to SEQ ID NO: 12. The polypeptide comprises SEQ ID NO: 13 or at least 80% sequence identity to SEQ ID NO: 13. The polypeptide comprises SEQ ID NO: 14 or at least 80% sequence identity to SEQ ID NO: 14. The polypeptide comprises SEQ ID NO: 15 or at least 80% sequence identity to SEQ ID NO: 15. The polypeptide comprises SEQ ID NO: 16 or at least 80% sequence identity to SEQ ID NO: 16. The polypeptide comprises SEQ ID NO: 17 or at least 80% sequence identity to SEQ ID NO: 17.

In one embodiment, the polypeptide sequence is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In one embodiment, the polypeptide is not a polypeptide of SEQ ID NO: 3.

In one embodiment, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3. In one embodiment, the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3, but the polypeptide is SEQ ID NO:3 It is not a polypeptide having the amino acid sequence of

In another embodiment, the polypeptide comprises amino acid residues 1-121 of SEQ ID NO:3 and amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 of the polypeptide sequence are listed SEQ ID NO: at least 1 compared to 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, or a range of values thereof) amino acid substitutions, additions, insertions, or deletions.

In another embodiment, the polypeptide sequence of a polypeptide having sweetness modulating activity encoded by a polynucleotide is a polypeptide comprising amino acid residues 1-121 of SEQ ID NO: 3, and amino acid residues 12-16, 33- 38, 41-44, 68-72, or 101-109 has at least one amino acid substitution, addition, insertion or deletion compared to SEQ ID NO: 3 of the listed residues, wherein the polypeptide is SEQ ID NO: 3 have at least 80% sequence identity.

In another embodiment, the invention provides SEQ ID NO: 3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO fused to a heterologous signal peptide or transit peptide. :12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 at least 80% (e.g., at least 81%, at least 82%, At least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% %, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity; The term "consisting essentially of" means a non-caking agent, filler, stabilizer (heat stabilizer, etc.), bulking agent (e.g., maltodextrose, acacia gum, etc.) that is not essential to, and does not significantly affect the function or activity of the product. It means that it contains ingredients that do not.

In another aspect, the present invention provides SEQ ID NO: 3 (or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 , SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17) at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%) %, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, SEQ ID NO 3 (or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO) having at least 97%, at least 98% or at least 99% sequence identity :12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17) containing at least one substitution modification, or essentially It consists of, or comprises a polypeptide having a sweet taste control activity consisting of. In some embodiments, a polypeptide of the invention has a position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to the corresponding amino acid of SEQ ID NO:3 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 , or 1 to 24 amino acid substitutions at 121. The term "consisting essentially of" means a non-caking agent, filler, stabilizer (heat stabilizer, etc.), bulking agent (e.g., maltodextrose, acacia gum, etc.) that is not essential to, and does not significantly affect the function or activity of the product. It means that it contains ingredients that do not.

In certain embodiments, the polypeptide sequence of a polypeptide having sweetness modulating activity encoded by a polynucleotide is SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75 It contains the amino acid sequence of

SEQ ID NO: 71 (consensus sequence 1) is at positions 3, 11-16, 26, 33, 34, 36-38, 41-43, 51, 57, 66, 68-72, 85, 86, 89, 97, Corresponds to SEQ ID NO: 3 except that 101-110, 117, and 120 are arbitrary amino acids. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 71.

SEQ ID NO: 72 (consensus sequence 2) is at position 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, Corresponds to SEQ ID NO: 3 except that 101-103, 105-110, 117, and 120 are any amino acids (i.e., at positions 34, 36, 42, 71 and 104 of SEQ ID NO: 3). proline is retained). The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 72.

SEQ ID NO: 73 (consensus sequence 3) and SEQ ID NO: 74 (consensus sequence 4) are positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70 , 72, 85, 86, 89, 97, 101-103, 105-110, 117, and 120 correspond to SEQ ID NO: 3 except that they may contain conservative modifications as described herein. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 73. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 74.

SEQ ID NO: 75 (consensus sequence 5) has positions 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117, and 120 as described herein. Corresponds to SEQ ID NO: 3 except that it may contain conservative modifications. The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 75.

In certain embodiments, the polypeptide sequence of a polypeptide having sweetness modulating activity is residues 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110, 117 of SEQ ID NO: 3 SEQ ID NO with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16) modifications at , and 120 : contains 3. Exemplary modifications to SEQ ID NO: 3 (for polypeptides) are presented herein (Example 8; see Table 3). For example, the polypeptide sequence of a polypeptide having sweetness regulating activity is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO; 64, SEQ ID NO: 66, or SEQ ID NO: 68 or SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44 , SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO; 64, SEQ ID NO: 66, or SEQ ID NO: 68 and at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.

The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 24 (D3E) or SEQ ID NO: 24. and at least 80% sequence identity with the polypeptide SEQ ID NO: 26 (K11R) or SEQ ID NO: 26 of a polypeptide having sweetness regulating activity. The polypeptide sequence of a polypeptide having sweetness regulating activity includes at least 80% sequence identity to SEQ ID NO: 30 (K26R) or SEQ ID NO: 30. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 38 (K51R) or SEQ ID NO: 38. The sweetness modulating activity comprises SEQ ID NO: 42 (R57K) or at least 80% sequence identity with SEQ ID NO: 42. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity to SEQ ID NO: 44 (R66K) or SEQ ID NO: 44. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 46 (D69E) or SEQ ID NO: 46. The polypeptide sequence activity of a polypeptide having sweet taste regulatory activity comprises at least 80% sequence identity with SEQ ID NO: 50 (D85E) or SEQ ID NO: 50. The polypeptide sequence of a polypeptide having sweetness modulating activity comprises at least 80% sequence identity with SEQ ID NO: 52 (E86D) or SEQ ID NO: 52. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity to SEQ ID NO: 54 (E89D) or SEQ ID NO: 54. Polypeptide Sequence of Polypeptide A polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 58 (D97E) or SEQ ID NO: 58. The polypeptide sequence of a polypeptide having sweetness regulating activity includes at least 80% sequence identity with SEQ ID NO: 60 (K103R) or SEQ ID NO: 60. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 62 (R106K) or SEQ ID NO: 62. The polypeptide sequence regulatory activity of a polypeptide having sweet taste regulatory activity comprises at least 80% sequence identity with SEQ ID NO: 64 (R110K) or SEQ ID NO: 64. The polypeptide sequence of a polypeptide having sweetness regulating activity comprises at least 80% sequence identity with SEQ ID NO: 66 (E117D) or SEQ ID NO: 66. The polypeptide sequence of a polypeptide having sweet taste regulatory activity comprises at least 80% sequence identity with SEQ ID NO: 68 (K120R) or SEQ ID NO: 68.

In another embodiment, a polynucleotide encoding a polypeptide having sweet taste regulating activity is SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67 or SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43 , SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67 and at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity.

The polynucleotide comprises SEQ ID NO: 23 (corresponding to D3E) or at least 80% sequence identity to SEQ ID NO: 23. The polynucleotide comprises SEQ ID NO: 25 (corresponding to K11R) or at least 80% sequence identity to SEQ ID NO: 25. The polynucleotide comprises SEQ ID NO: 29 (corresponding to K26R) or at least 80% sequence identity to SEQ ID NO: 29. The polynucleotide comprises SEQ ID NO: 37 (corresponding to K51R) or at least 80% sequence identity to SEQ ID NO: 37. The polynucleotide comprises SEQ ID NO: 41 (corresponding to R57K) or at least 80% sequence identity to SEQ ID NO: 41. The polynucleotide comprises SEQ ID NO: 43 (corresponding to R66K) or at least 80% sequence identity to SEQ ID NO: 43. The polynucleotide comprises SEQ ID NO: 45 (corresponding to D69E) or at least 80% sequence identity to SEQ ID NO: 45. The polynucleotide comprises SEQ ID NO: 49 (D85E) or at least 80% sequence identity to SEQ ID NO: 49. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 51 (corresponding to E86D) or SEQ ID NO: 51. The polynucleotide comprises SEQ ID NO: 53 (corresponding to E89D) or at least 80% sequence identity to SEQ ID NO: 53. The polynucleotide comprises SEQ ID NO: 57 (corresponding to D97E) or at least 80% sequence identity to SEQ ID NO: 57. The polynucleotide comprises SEQ ID NO: 59 (corresponding to K103R) or at least 80% sequence identity to SEQ ID NO: 59. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 61 (corresponding to R106K) or SEQ ID NO: 61. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 63 (R110K) or SEQ ID NO: 63. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 65 (E117D) or SEQ ID NO: 65. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 67 (K120R) or SEQ ID NO: 67.

In one embodiment, the polynucleotide comprises SEQ ID NO: 20 corresponding to the coding sequence of His-tagged mycodulcein in E. coli, wherein residues 364-381 correspond to an optional His-tag sequence. SEQ ID NO: 20 is codon-optimized for expression in E. coli. In another embodiment, the polynucleotide comprises SEQ ID NO: 22, which corresponds to the coding sequence of His-tagged mycodulcein from S. cerevisiae, wherein residues 364-381 are an optional His-tag corresponding to the sequence). SEQ ID NO: 22 is codon optimized for expression in S. cerevisiae. The corresponding polypeptide of SEQ ID NO: 21 corresponds to His-tagged mycodulcein protein, wherein residues 122-127 correspond to any His tag sequence, and this polypeptide sequence is found in E. coli and S. cerevisiae. Same for expression. Accordingly, the present invention also provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 21.

The present invention also relates to the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 48, or SEQ ID NO: 56; or at least 80% (e.g., SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 48, or SEQ ID NO: 56) 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99%) sequence identity.

The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 28 (R20K) or SEQ ID NO: 28. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 32 (E35D) or SEQ ID NO: 32. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 34 (K44R) or SEQ ID NO: 34. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 36 (D46E) or SEQ ID NO: 36. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 40 (D52E) or SEQ ID NO: 40. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 48 (R75K) or SEQ ID NO: 48. The polypeptide comprises at least 80% sequence identity to SEQ ID NO: 56 (D94E) or SEQ ID NO: 56.

In another embodiment, the polynucleotide is SEQ ID NO: 27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55 , or at least 80% of SEQ ID NO: 27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO:55 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97 %, 98% or 99%) sequence identity.

The polynucleotide comprises SEQ ID NO: 27 (corresponding to R20K) or at least 80% sequence identity to SEQ ID NO: 27. The polynucleotide comprises SEQ ID NO: 31 (corresponding to E35D) or at least 80% sequence identity to SEQ ID NO: 31. The polynucleotide comprises SEQ ID NO: 33 (corresponding to K44R) or at least 80% sequence identity to SEQ ID NO: 33. The polynucleotide comprises at least 80% sequence identity to SEQ ID NO: 35 (corresponding to D46E) or SEQ ID NO: 35. The polynucleotide comprises SEQ ID NO: 39 (corresponding to D52E) or at least 80% sequence identity to SEQ ID NO: 39. The polynucleotide comprises SEQ ID NO: 47 (corresponding to R75K) or at least 80% sequence identity to SEQ ID NO: 47. The polynucleotide comprises SEQ ID NO: 55 (corresponding to D94E) or at least 80% sequence identity to SEQ ID NO: 55.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids.

A polynucleotide or polypeptide may be naturally occurring or non-naturally occurring (eg, synthetic, recombinant, modified and/or variant product). In one embodiment, the naturally occurring or non-naturally occurring product is isolated or purified.

In one embodiment, the term "isolated" includes a product that has been removed from a biological environment (eg, cells, tissues, culture media, bodily fluids, etc.) or whose purity has been increased to some extent (eg, separated from a synthetic medium). Thus, an isolated product may be synthetic or produced naturally.

As used herein, the term "isolated" when referring to a nucleic acid or polypeptide means a state of purification or concentration different from that which naturally occurs. (1) purification from other naturally occurring related structures or compounds, or (2) greater degree of purification or enrichment than naturally occurring, including association with structures or compounds not normally associated in the body. within the meaning of "isolated" as used in

As used herein, "recombinant" means a polynucleotide synthesized or otherwise engineered in vitro (eg, a "recombinant polynucleotide"), a method of using a recombinant polynucleotide to produce a gene product in a cell or other biological system. , or a polypeptide encoded by a recombinant polynucleotide ("recombinant protein"). "Recombinant means" also refers to the expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using the primers of the invention, eg various coding regions into an expression cassette or vector for inducible or constitutive expression, or domains, or linking of nucleic acids with promoter sequences from different sources. .

A “modified” or “variant” product refers to a product (eg, a polynucleotide or polypeptide) that has been altered from its original (eg, naturally occurring) structure. As described herein, variants include polynucleotides or polypeptides having one or more changes to the nucleic acid or amino acid sequence, respectively. Modifications include modifications to nucleic acid or amino acid sequences, including additions, deletions, insertions and substitutions. A modified or modified product may also include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as conjugation with a labeled component relative to the original structure.

The terms "amplifying" and "amplification" as used herein refer to the use of any suitable amplification methodology for generating or detecting recombinant or naturally expressed nucleic acids, as described in detail below. For example, the present invention provides a naturally expressed (eg, genomic or mRNA) or recombinant (eg, cDNA) nucleic acid (eg, a taste stimulus-binding sequence of the present invention) in vivo or in vitro (eg, a taste stimulus-binding sequence of the present invention). , by polymerase chain reaction, PCR) and reagents (eg, specific degenerate oligonucleotide primer pairs) for amplification.

The term "library" refers to a mixture of different nucleic acid or polypeptide molecules, such as a library of recombinantly produced Myd-related polynucleotides produced by nucleic acid amplification with degenerate primer pairs or an isolated collection of vectors comprising amplified ligand-binding domains. , or a mixture of cells each randomly transfected with at least one vector encoding MYD.

As used herein, a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through the formation of hydrogen bonds. . As used herein, a probe may contain natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases of the probe may be linked by a bond other than a phosphodiester bond as long as it does not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid whose constituent bases are linked by peptide bonds other than phosphodiester bonds. One skilled in the art will appreciate that a probe may bind to a target sequence that lacks complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. The probe is optionally labeled directly, such as an isotope, chromophore, luminophore, chromophore, or indirectly, such as biotin to which the streptavidin complex may bind later. By assaying for the presence or absence of a probe, the presence or absence of a selected sequence or subsequence can be detected.

The term "heterologous" when used in reference to a portion of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in nature in identical relation to one another. For example, nucleic acids are generally produced recombinantly with two or more sequences from unrelated genes aligned to create a new functional nucleic acid, eg, a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that are not found in nature in the same relationship to each other (eg, a fusion protein).

A "promoter" is defined as an arrangement of nucleic acid sequences that directs the transcription of a nucleic acid. Promoters as used herein include the necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. Promoters also optionally include distal enhancer or repressor elements, which may be located thousands of base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. A “regulatory” promoter is a promoter that is activated under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter or transcription factor binding site array) and a second nucleic acid sequence, wherein the expression control sequence is used for transcription of a nucleic acid corresponding to the second sequence. instruct

The term "MYD family" refers to at least about 35 to 50% amino acid sequence identity to SEQ ID NO:3 over a window of about 25 amino acids, optionally 50-100 amino acids, optionally about 60, 75, 80, 85, 90, polymorphic variants, including natural alleles, mutations, alleles and interspecies homologues that encode polypeptides having 95, 96, 97, 98, or 99% amino acid sequence identity.

The term “expression vector” or “expression cassette” refers to a nucleic acid sequence of the present invention either constitutively or inducibly in vitro or in vivo in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cells. Refers to any recombinant expression system for expressing. This term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. An expression system may or may not be self-replicating. That is, only transient expression can be induced in cells. The term includes a recombinant expression "cassette" that contains only the minimal elements required for transcription of a recombinant nucleic acid.

"Host cell" means a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, HEK-293, etc., eg cultured cells, explants and in vivo cells. .

In one embodiment, the host cell is Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succinisiproducens, rhizo Bium atli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostri Dium acetobutylicum, Pseudomonas fluorescens, Pseudomonas putida, Saccharomyces cerevisiae, Shizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marcianus, Aspergillus tereus , Aspergillus niger, Pichia pastoris, Rhizopus arhijuus, Rhizopus oryzae, Yarrowia lipolytica, Candida albicans, Isachenchia orientalis, Sheffersomyces stipitis, Yarrow Weia lipolytica, Ogataea polymorpha, Papia rhodozyme, Candida utilis, Arsula adeninivorans, Devariomyces hansenii, Devariomyces polymorphus and Schwanniomyces occidentalis selected from the group consisting of

In another aspect, the host cell is selected from the group consisting of gram-positive asporogenic bacteria, gram-positive spore-forming bacteria, gram-negative bacteria, yeasts, and protists/algae.

Non-limiting examples of Gram-positive asporogenic bacteria include Bifidobacterium adelessontis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium brev, Bifidobacterium longum, Carnobacterium Diversens, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amyloborus, Lactobacillus animalis, Lactobacillus alimentarius, Lactobacillus avirie, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus cholinoides, Lactobacillus corniniformis, Lactobacillus crispatus, Lactobacillus curba Tooth, Lactobacillus delbruecki, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus parsiminis, Lactobacillus fermentum, Lactobacillus galinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hill Gardi, Lactobacillus johnsonni, Lactobacillus kepyranofaciens, Lactobacillus kepiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paraparaginis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus san Francissensis, Lactococcus lactis, Leuconostoc Citrium, Leuconostoc lactis, Leuconostoc mesenteroids, Leuconostoc pseudomesenteroides, Microbacterium imperial, Oenococcus oeni, Pasturia nishizawaae, Pediococcus acidiact, Pediococcus parbulus, Pediococcus pentosaceus, Propionibacterium acidipropioni, Propionibacterium proidenreichii, and Streptococcus thermophiles.

Non-limiting examples of Gram-positive spore-forming bacteria include Bacillus amyloliquefaciens, Bacillus atropheus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus plexus, Bacillus fusiformis, Bacillus lentus, Bacillus Richeniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus smishii, Bacillus subtilis, Bacillus valesmortis, Bacillus bellegensis, Geobacillus stearomophilus, Paenibacillus illinois Sensis, and Phageobacillus thermoglucosidacius. Non-limiting examples of Gram-negative bacteria include Cupriavidus necator, Gluconobacter oxydans, Comagata Abacter sucrofermentans, and Xanthomonas campestris.

Non-limiting examples of yeast include Candida cilindracea, Devariomyces hansenii, Hanseniaspora ubarum, Kluyveromyces lactis, Kluyveromyces marcianus, Comagataella pastoris, Comagataella papi, Lind Nera jardiniii, Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus, Shizoscharomyces pombe, Wickerhamomyces anomalus, Xantho Philomyces dendrohaus, Yarrowia lipolytica and Higosaccharomyces lucii.

Non-limiting examples of protists/algae include Aurantiochytrium limacinum, Euglena Graciformis, and Tetraselmis twit.

The Myd proteins described herein also include "analogues" or "conservative variants" and "mimics" ("peptidomimetics") having structures and activities substantially corresponding to the exemplary sequences. Thus, the term "conservative variant" or "analog" or "mimetic" refers to a polypeptide having a modified amino acid sequence, the modification(s) being the structure and/or structure of the polypeptide (conservative variant) as defined herein Does not substantially change activity. These include conservative modifications of the amino acid sequence, i.e. amino acid substitutions, additions or deletions of residues not critical to protein activity, or residues with similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.). Substitutions of amino acids are included, provided that substitutions of key amino acids do not substantially alter structure and/or activity.

More specifically, "conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence or, when the nucleic acid does not encode an amino acid sequence, essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode a given protein.

For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Thus, at every position where an alanine is designated by a codon, the codon can be changed to any corresponding codon described without altering the encoded polypeptide.

Such nucleic acid variances are "silent variances", which are a type of conservatively modified variance. Every nucleic acid sequence herein that encodes a polypeptide also describes all possible silent variations of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to create functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is implied in each described sequence.

Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline for selecting conservative substitutions includes (original residues followed by exemplary substitutions): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. Another exemplary guideline uses six groups, each containing amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (I); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W); (See, eg, Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). Another exemplary guideline uses the following six groups, where proline is unique: 1) Gly (G), Ala (A), Val (V), Leu (L), Ile (I); 2) Ser(S), Cys(C), Thr(T), Met(M); 3) Pro (P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6) Asp (D), Glu (E), Gln (N). Those skilled in the art will understand that the substitutions identified above are not the only possible conservative substitutions. For example, for some purposes all charged amino acids, whether positive or negative, can be considered conservative substitutions for each other. Individual substitutions, deletions or additions that alter, add or delete single amino acids or small amounts of amino acids in the coded sequence may also be considered "conservatively modifying variations". One skilled in the art will be familiar with codon selection in a given host expressing the protein in question.

The terms "mimetic" and "peptidomimetic" refer to synthetic chemical compounds having substantially the same structural and/or functional characteristics of a polypeptide of the invention, e.g., a translocation domain, ligand-binding domain or chimeric receptor. A mimetic can be composed entirely of synthetic, non-natural analogs of amino acids, or can be chimeric molecules of natural peptide amino acid portions and non-natural analogs of amino acids. The mimetibody may also contain any amount of natural amino acid conservative substitutions, so long as such substitutions do not substantially alter the structure and/or activity of the mimetibody.

As with polypeptides of the invention that are conservative variants, routine experimentation will determine whether the mimic is within the scope of the invention, i.e., its structure and/or function is not substantially altered. Polypeptide mimetic compositions may contain any combination of non-natural structural elements, which generally fall into three structural groups: a) residue linkages other than natural amide linkages ("peptide linkages") linkages; b) unnatural residues in place of naturally occurring amino acid residues; or c) moieties that induce secondary structure mimicry, i.e. induce or stabilize secondary structure, e.g., beta turns, gamma turns, beta sheets, alpha helices, etc. A polypeptide may be characterized as a mimetic when all or some of its residues are linked by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be combined with peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, difunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC ) or N,N'-diisopropylcarbodiimide (DIC). An alternative linking group to the traditional amide bond ("peptide bond") linkage is, for example, ketomethylene (eg, -C(O)--CH ₂ - for -C(O)--NH--) -), aminomethylene (CH ₂ -NH), ethylene, olefin (CH=CH), ether (CH ₂ -O), thioether (CH-S), tetrazole (CN ₄ ), thiazole, retroamide, thioamides or esters (see, eg, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY (1983)). Polypeptides may also be characterized as mimetics by containing all or part of non-natural residues in place of naturally occurring amino acid residues; Non-natural residues are well described in the scientific and patent literature. Phyre2 is a collection of tools available on the web for predicting and analyzing protein structure, function and mutations.

Protein folding analysis is performed using tools including PHYRE Protein Homology/analogY Recognition Engine V 2.0 and JPred, a protein secondary structure prediction server. These tools reveal that SEQ ID NO:3 predicts a β sheet-predominant globular protein with a substantial β-sheet section and some short α-helical sections. Specifically, the Jpred tool uses putative β sheets at approximately residues 5-11, 16-19, 28-29, 39-40, 61-67, 71-77 and 98-101 of SEQ ID NO:3 and approximately residues 20- 25, 45–55, predicts an α-helix at 111–119; The PHYRE tool uses the β sheet at approximately residues 5-12, 17-32, 38-40, 45-57, 62-66, 73-81, 98-104 of SEQ ID NO:3 and at approximately residues 84-89 and 116- 118 predicts an α-helix. See Figure 1.

Examples of conservatively modified variations of the Myd1 protein structure can be derived using homology modeling algorithms: SWISS-MODEL, PHYRE2.0 and Jpred to identify sequence-based consensus loop regions as known in the art. For example, Pechmann, S. & Frydman, J. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks. See PLoS Computational Biology 10, e1003674 (2014).

Alternative protein sequences with putatively similar structure and function to Myd1 are provided herein as SEQ ID NO:8 to SEQ ID NO:17. To derive SEQ ID NO:8 to SEQ ID NO:17, the common loop region was chosen as the site of mutation because most insertions and deletions are usually found in the region between secondary structural elements, and in the entire fold of the protein. It can be more easily accommodated without major distortion. The core of this protein has a higher degree of sequence conservation as found within 4JOX.

Of the 29 possible amino acid positions within the common loop region, 12 amino acids were replaced using conservative replacements. When selected for mutation, wild-type amino acids were given with equal probability among conservative amino acid residues. (Gly can be replaced with Ala, Cys, Asp, Glu, Arg with equal 20% probability).

Specific regions of the MYD/Myd nucleotide and amino acid sequences can be used to identify polymorphic variants, interspecies homologs and alleles of Myd family members. Such identification can be made in vitro, for example, under stringent hybridization conditions or PCR (eg, using primers encoding the Myd sequences identified herein) or using sequence information in a computer system for comparison to other nucleotide sequences. . Different alleles of the MYD gene within a single species population would also be useful in determining whether differences in allelic sequence correlate with differences in taste perception among members of the population. Classical PCR type amplification and cloning techniques are useful, for example, to isolate orthologs where degenerate primers are sufficient to detect related genes across species.

For example, primers designed using the sequences disclosed herein can be used to amplify and clone MYD-related genes from different fungal genomes. In contrast, genes within a single species associated with MYD are best identified using sequence pattern recognition software to find related sequences. Generally, identification of polymorphic variants and alleles of MYD family members can be made by comparing amino acid sequences of at least about 25 amino acids, for example 50-100 amino acids. Amino acid identities of at least 35 to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99% or more, are generally considered to be proteins of the polymorphic variant, cross-species homologue or MYD family. Prove that it is an allele of the member. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that specifically bind to Myd polypeptides or conserved regions thereof can also be used to identify alleles, interspecies homologues and polymorphic variants.

Nucleotide and amino acid sequence information for MYD family members can also be used to construct models of sweet regulatory polypeptides and how they interact with sweet taste receptors in a computer system and the same computer system model. The sweet receptor consists of a heterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptor member 3 (T1R3). This model can subsequently be used to identify variations and mutations of Myd that can increase activation of the receptor, and to identify more active versions of Myd.

A variety of conservative mutations and substitutions are considered within the scope of this invention. It is within the level of skill in the art to perform amino acid substitutions using known protocols of recombinant genetic technology, including, for example, PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription. there is. The variants can then be screened for taste receptor agonist functional activity.

In one embodiment, a hybrid protein-coding sequence comprising a nucleic acid encoding a Myds fusion protein can be constructed. These nucleic acid sequences can be operably linked to transcriptional or translational control elements such as transcriptional and translational initiation sequences, promoters and enhancers, transcriptional and translational terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. . The fusion protein may include a C-terminal or N-terminal translocated sequence. Fusion proteins may also include additional elements, for example for protein detection, purification or other applications. Detection and purification facilitating domains include, for example, metal chelating peptides such as polyhistidine tracts, histidine-tryptophan modules or other domains capable of purification from immobilized metals; maltose binding protein; Protein A domains that can be purified from immobilized immunoglobulins; or the domain used in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.).

In one embodiment, the fusion protein includes a peptide or protein tag (eg, for protein purification or detection). Peptide/protein tags are known in the art as described by Johnson, “Protein/Peptide Tags,” DOI //dx.doi.org/10.13070/mm.en.2.116. Green fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine, glutathione-S-transferase (GST), HA, V5, ABDz1-tag, adenylate kinase (AK-tag), BC2-tag, calmodulin-binding peptide, CusF, Fc , Fh8, Halo tag, heparin binding peptide (HB-tag), ketosteroid isomerase (KSI), maltose binding protein (MBP), thioredoxin, PA (NZ-1), Poly-Arg, Poly-Lys, S-tag, SBP/streptavidin binding peptide, SNAP, Strep-II (Twin-Strep) and SUMO/SUMO2.

An affinity tag is a type of protein tag attached to a protein so that the protein can be purified from a crude biological source using affinity techniques. Affinity tags are described in Kimple et al. Curr Protoc Protein Sci.; 73: Unit-9.9. It is known in the art as described in doi:10.1002/0471140864.ps0909s73. These include polyhistidine, GST, MBP, calmodulin-binding peptides, intein chitin binding domains, streptavidin/biotin based tags and His-patch thioredoxins (ThioFusion). Affinity tags include small (eg, less than 20 amino acid residues) or large affinity tags. Examples of small affinity tags include His, FLAG, Strep II and S-peptide, examples of large affinity tags include MBP, GST, cellulose binding domain, calmodulin binding peptide and His-patch thioredoxin do.

Affinity tags include epitope tags and reporter tags. Reporter tags serve as reporters of protein expression and protein-protein interactions. Reporter tags include, but are not limited to, enzymes such as β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP). don't

Epitope tags include FLAG, hemagglutinin (HA), c-myc, T7 and Glu-Glu, which are used for detection of fusion proteins in vitro and in cell culture. Their short linear recognition motifs have little effect on the properties of the protein of interest and are usually highly specific for each primary antibody. When using an anti-myc antibody, specificity can be increased by using an enzyme-linked secondary antibody to detect the conjugated anti-myc primary antibody instead of using the HRP- or AP-anti-myc conjugate alone. there is.

Tags can be at both ends of the target protein. Some epitope tags, such as FLAG, are often used together or with other tags, as in the structures of His-Myc and His-V5, to augment a desired function.

Tandem affinity purification (TAP) is a dual-affinity purification method based on the fusion of two affinity tags to a protein of interest, which allows purification of the tagged protein and isolation of protein complexes that interact with the protein of interest. let it The use of TAP is included in the present invention.

In one embodiment, the fusion protein comprises a histidine tag comprising 2-10 (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10) histidine residues. For example, a histidine tag may contain 6 histidine residues.

A cleavable linker sequence between the translocation domain (for efficient plasma membrane expression) and the remainder of the newly translated polypeptide, such as factor Xa (see, e.g., Ottavi, Biochimie 80:289-293 (1998)), a subtilisin protease recognition motif (e.g. , Polyak, Protein Eng. 10:615-619 (1997)); Inclusion of enterokinase (Invitrogen, San Diego, Calif.) and the like may be useful to facilitate purification. For example, one construct comprises a nucleic acid sequence linked to six histidine residues followed by a thioredoxin, an enterokinase cleavage site (see, eg, Williams, Biochemistry 34:1787-1797 (1995)) and a C-terminal translocation domain. It may contain a polypeptide that encodes. The histidine residue facilitates detection and purification while the enterokinase cleavage site provides a means to purify the desired protein(s) from the rest of the fusion protein. Vectors encoding fusion proteins and techniques for application of fusion proteins are described in the scientific and patent literature, eg Kroll, DNA Cell. Biol. 12:441-53 (1993).

A fusion protein may contain one or more linkers (eg, flexible linkers, rigid linkers, and in vivo cleavable linkers). Moreover, in addition to their primary role in linking functional domains together (as in flexible and rigid linkers) or releasing free functional domains in vivo (as in in vivo cleavable linkers), linkers can be used in many other ways for the production of fusion proteins. benefits such as improved biological activity, increased yield of expression, achievement of desirable pharmacokinetic profiles, and the like. Linkers are known in the art (see, eg, Chen et al., Adv Drug Deliv Rev. 65(10): 1357-1369 (2013)).

Flexible linkers are used when the linked domains require some degree of movement or interaction. They are usually composed of small non-polar (eg Gly) or polar (eg Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows mobility of the linking functional domains. Incorporation of Ser or Thr can form hydrogen bonds with water molecules to maintain the stability of the linker in aqueous solution, thereby reducing undesirable interactions between the linker and the protein moiety.

The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linkers). An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 69). Adjusting the copy number “n” can optimize the length of this GS linker to properly separate the functional domains or maintain the necessary inter-domain interactions. Besides the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility and polar amino acids such as Lys and Glu to enhance solubility.

A tight linker maintains a fixed distance between domains and maintains their independent function. Examples of tight linkers include an alpha helix-forming linker having a sequence of (EAAAK)n (SEQ ID NO: 70) and a linker having a Pro-rich sequence (XP)n, where X is any amino acid, preferably represents Ala, Lys or Glu. , .

Polypeptides of the present invention may also contain a signal peptide (i.e., a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide), which is responsible for most newly synthesized proteins directed to the secretory pathway. It is a short peptide present at the N-terminus or sometimes at the C-terminus. These proteins include proteins that are present inside certain organelles (endoplasmic reticulum, Golgi apparatus or endosomes), are secreted from cells, or are incorporated into most cell membranes. Exemplary signal peptides are known in the art and one skilled in the art will recognize how to select a particular signal peptide for use in the present invention.

As used herein, "at least 80% identity" with respect to an amino acid sequence or nucleotide sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% identity.

Examples of the "amino acid sequence modified by deletion, insertion, substitution or addition of one or more amino acids" herein include 1 or more to 30 or less, preferably 20 or less, more preferably 10 or less, still more preferably Less than 5 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range thereof) modified by deletion, insertion, substitution or addition of amino acids. Examples of "base sequence modified by deletion, insertion, substitution or addition of one or more nucleotides" in the present specification include 1 or more to 90 or less, preferably 60 or less, more preferably 30 or less, still more preferably Preferably 15 or less, more preferably 10 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or any of these range) of nucleotide sequences modified by deletion, insertion, substitution or addition.

For example, in sequence comparison, one sequence generally serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, sequence coordinates are designated as necessary, and sequence algorithm program parameters are specified. You can use default program parameters or specify alternate parameters, as described below for the BLASTN and BLASTP programs. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

As used herein, a "comparison window" refers to a segment of any one of a number of contiguous positions selected from the group consisting of 20 to 600, typically from about 50 to about 200, and more typically from about 100 to about 150. After the two sequences are optimally aligned, the sequence can be compared to a reference sequence at the same number of contiguous positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison is described, for example, in Smith & Waterman, Adv. Appl. Math. 2:482 (1981) by the local homology algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970) by the homology alignment algorithm, Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988) by the similarity search method, to a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by manual alignment and visual inspection (see, eg, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul at al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying short words of length W in the query sequence to identify high-scoring sequence pairs (HSPs). This word matches or satisfies some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighbor word score threshold (Altschul et al., Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410 ( 1990)). These initial neighbor word hits serve as seeds for starting the search to find longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. A cumulative score is calculated using, for nucleotide sequences, the parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a cumulative score is calculated using a scoring matrix. Word hit expansion in each direction stops when the cumulative alignment score falls a number X from the maximum achieved value; The cumulative score goes below zero due to the accumulation of one or more negative scoring residue alignments, or the end of the sequence has been reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to using a word length (W) of 11, an expected value (E) or 10, M=5, N=-4 and a comparison of the two strands. For amino acid sequences, the BLASTP program defaults to word length of 3, expected value (E) of 10, and BLOSUM62 scoring matrix alignment (B) of 50, expected value (E) of 10, M=5,N=-4, and two strands. use the comparison of

Another example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignments to generate multiple sequence alignments from groups of related sequences, displaying relationships and percent sequence identity. We also plot so-called "trees" or "dendograms" showing the clustering relationships used to generate the alignments (eg see Figure 2). PILEUP is from Feng & Doolittle, J Mol. Evol. 35:351-360 (1987) is simplified and used. The method used is similar to that described by Higgins & Sharp, CABIOS 5:151-153 (1989). This program can align up to 300 sequences, each up to 5,000 nucleotides or amino acids in length. The multiple alignment procedure starts with pairwise alignment of the two most similar sequences, which creates a cluster of two aligned sequences. These clusters are then aligned to the cluster of the next most related or ordered sequence. The two clusters of sequences are aligned by a simple extension of the pairwise alignment of the two individual sequences. Final alignment is achieved by a series of progressive pairwise alignments. Programs are run by specifying amino acid or nucleotide coordinates for specific sequences and regions of sequence comparison and specifying program parameters. PILEUP is used to compare the reference sequence to other test sequences to determine the percent sequence identity relationship using the default gap weight (3.00), default gap length weight (0.10), and weighted end gap parameters. PILEUP can be obtained from the GCG sequence analysis software package, eg version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)) and is used for conceptual translation of corresponding open reading frames encoded by genes. was derived by

Polynucleotides encoding the polypeptides of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of Myd. For example, a polynucleotide can be chemically synthesized based on the amino acid sequence of a polypeptide of the invention or a preprotein thereof. For chemical synthesis of polynucleotides, a nucleic acid consignment synthesis service (eg, provided by Medical & Biological Laboratories Co., Ltd., Genscript, etc.) can be used. In addition, the synthesized polynucleotide can be amplified by PCR, cloning, and the like.

Polypeptides of the invention can be produced, for example, by expressing a gene encoding a Myd polypeptide of the invention. Preferably, the Myd polypeptide of the present invention can be produced from a transformant into which a polynucleotide encoding the Myd polypeptide of the present invention has been introduced. For example, the Myd polypeptide of the present invention is introduced into a transformant by introducing a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the same into a host to obtain a transformant, culturing the transformant in an appropriate medium, and then introducing the transformant into the transformant. produced from polynucleotides encoding the Myd polypeptides of the present invention. The protein of the present invention can be obtained by isolating or purifying the produced Myd polypeptide from culture.

Accordingly, the present invention further provides polynucleotides encoding the Myd polypeptides of the present invention and vectors comprising the same. The present invention also provides a method for producing a transformant comprising introducing a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the polynucleotide into a host. The present invention also provides a transformant comprising a polynucleotide encoding the Myd polypeptide of the present invention or a transformant introduced from the outside of a cell with a vector containing the same. The present invention further provides a method for producing the Myd polypeptide of the present invention comprising culturing the transformant.

The invention also includes polynucleotides of the invention operably linked to heterologous regulatory elements. The present invention may include an expression cassette or vector comprising the polynucleotide of the present invention, and a host cell transformed with the vector of the present invention.

Alternatively, polynucleotides encoding the Myd polypeptides of the present invention can be generated by introducing mutations into polynucleotides synthesized according to procedures known mutagenesis methods such as ultraviolet irradiation and site-directed mutagenesis. By introducing a mutation into the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2 by a known method, expressing the obtained polynucleotide, examining the sweetness-modifying activity of the expressed protein, and then obtaining the desired sweetness-modifying activity. A polynucleotide encoding a polypeptide of the present invention can be obtained by selecting a polynucleotide encoding a protein.

Site-directed mutagenesis of polynucleotides can be performed by any method, such as, for example, inverse PCR and annealing (Muramatsu et al. edit., "Revised 4th edition New genetic engineering handbook", YODOSHA, p. 82-88 ). A variety of commercially available kits for site-directed mutagenesis, such as Stratagene's QuickChange II site-directed mutagenesis kit and QuickChange multiple site-directed mutagenesis kit, can be used as needed.

Examples of types of vectors comprising polynucleotides encoding the polypeptides of the present invention include, but are not limited to, vectors commonly used in gene cloning, such as plasmids, cosmids, phage, viruses, YACs and BACs. don't Examples of vectors include plasmids (e.g., DNA plasmids), yeast (e.g., Saccharomyces) and viral vectors, such as swviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, polioviruses, alphaviruses, baculoviruses, sindes vis viruses, plant viruses (eg Alphaflexiviridae or Potyviridae) and insect viruses (eg Baculoviridae).

Among these, plasmid vectors are preferable, and commercially available plasmid vectors for protein expression, such as pUC19, pUC118, pUC119, and pBR322 (all manufactured by TAKARA BIO INC.) can be used.

A vector may comprise a replication initiation region or a DNA region comprising the origin of replication of DNA. Alternatively, a regulatory sequence such as a promoter region that initiates transcription of a gene, a terminator region that secretes an expressed protein out of the cell, or a secretion signal region is a polynucleotide encoding a polypeptide of the present invention in a vector (i.e., a polynucleotide of the present invention). MYD gene) can be operably linked upstream. As used herein, genes and regulatory sequences that are “operably similar” refer to conditions in which the genes and regulatory regions are located so that the gene can be expressed under control by the regulatory regions.

The types of control sequences such as promoter regions, terminators and secretion signal regions are not particularly limited, and generally used promoters and secretion signal sequences may be appropriately selected and used depending on the host into which the sequences are introduced. For example, a preferred example of a regulatory sequence that can be introduced into the vector of the present invention includes the cbh1 promoter sequence from Trocherdoma reesei (Curr, Genet, 1995, 28(1):71-79).

Alternatively, a marker gene (e.g., a gene for resistance to agents such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector will be properly introduced can be further introduced into the vector of the present invention. . Alternatively, when an auxotrophic strain is used as a host, a gene encoding a synthesizing enzyme of a required nutrient can be introduced into the vector as a marker gene. Alternatively, when using a selective medium that requires a specific metabolism for growth, a metabolism-related gene can be introduced into the vector as a marker gene. An example of such a metabolism-related gene is an acetamidase gene for using acetamide as a nitrogen source.

The linkage between the polynucleotide encoding the Myd polypeptide of the present invention, the regulatory sequence and the marker gene can be performed by a method known in the art such as SOE (splicing byoverlap extension)-PCR (Gene, 1989, 77: 61-68). can Procedures for introducing linked fragments into vectors are known in the art.

Examples of the host of the transformant into which the vector is introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and bacteria belonging to the genus Bacillus (eg, Bacillus subtilis or variants thereof) are preferred. Examples of Bacillus subtilis variants include J. Biosci. Bioeng., 2007, 104 (2): protease 9 double deficient strain KA8AX described in 135-143, and Biotechnol. Lett., 2011, 33 (9): 1847-1852, a mutant DBPA strain from a protease 8 double deficient strain, of which protein folding efficiency is improved. Examples of filamentous fungi include Trichoderma, Aspergillus and Rhizopus. Also suitable expression hosts are, for example, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Shizoscharomyces pombe, Kluyveromyces lactis. In another aspect, the invention includes a host cell comprising one or more expression cassettes described herein operably linked to control elements compatible with expression in the cell. Cells include, for example, mammalian cells (eg BHK, VERO, HT1080, 293, RD, COS-7 or CHO cells), insect cells (eg Trichoplusia ni(Tn5) or Sf9), bacterial cells, plant cells or yeast cells. can be

Recombinantly expressed polypeptides from Myd-encoding expression cassettes are usually isolated from lysed cells or culture medium. Purification can be performed by methods known in the art including salt fractionation, ion exchange chromatography, gel filtration, size exclusion chromatography, size fractionation and affinity chromatography. For example, immunoaffinity chromatography can be used using antibodies generated based on the Gag antigen.

The present invention provides sweetness control comprising the steps of (a) obtaining a composition comprising a polypeptide, and (b) purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). A method for purifying a polypeptide having an activity is provided. In one embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ and an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another embodiment, the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (a) the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID comprising at least one substitution modification to a polypeptide sequence selected from the group consisting of NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is the polypeptide of SEQ ID NO:3 or (b) wherein the polypeptide further comprises a histidine tag, wherein the polypeptide has sweetness modulating activity.

One skilled in the art is familiar with the purification techniques of hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), including the selection of appropriate columns, buffers and elution solutions. Exemplary HIC and SEC purification techniques are described in Example 11 herein. In an exemplary embodiment, the purification rate of the polypeptide after purification by HIC and SEC is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any range of values.

The invention also contemplates transgenic plants comprising heterologous polynucleotides and/or heterologous polypeptides of the invention as described herein. Plants have altered phenotypes due to the expression of heterologous nucleic acid sequences. The altered phenotype may include an increased sweetness in all plant parts including fruits. A transgenic plant may comprise an expression cassette as defined herein as part of the plant, which cassette was introduced by transforming the plant with the vector of the present invention. Such expression cassettes contain regulatory sequences for expression of heterologous coding sequences in plants, including plant-expressible promoters and terminators. A transgenic plant can be any type of plant capable of expressing a heterologous nucleic acid sequence described herein. The term “plant” includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, and plant cells and their progeny. The plant classes that can be used in the methods of the present invention are as broad as the higher plant classes suitable for the transformation technique, which generally include both monocotyledonous plants (dicots) and dicotyledonous plants. This includes plants with various levels of polyploidy, including polyploid, diploid and haploid. For example, transgenic plants can be apples or strawberries.

Techniques for transforming a wide variety of plant species are well known in the art and described in the technical and scientific literature. For example, Weising et al. (1988) Ann. Rev. Genet., 22:421-477 and Joung et al. (2015) “Plant Transformation Methods and Applications,” in Current Technology in Plant Molecular Breeding, (Koh et al., eds) Springer Dordrecht Heidelberg New York London, Chapter 9, pages 297-344. Any method known in the art for transformation of plant cells, including plant protoplasts or plant tissues, can be used for plant transformation. Specific methods for plant transformation include the ballistic method (gene gun), electroporation, microinjection, protoplast fusion and Agrobacterium-mediated transformation, among others. For example, Agrobacterium-mediated transformation can use binary vectors that replicate in Escherichia coli and Agrobacterium tumefaciens or other Agrobacterium strains. A variety of such binary vectors are known in the art and can be used to introduce heterologous polynucleotides into plant cells and plant tissues. Plant expression vectors comprising regulatory sequences for the expression of heterologous coding sequences comprising plant-expressible promoter sequences and other plant regulatory sequences in plant cells and plant tissues are known in the art and express polypeptides as described herein. It can be used to transform plants to .

A variety of plant-expressible promoters are known in the art and are available for use in heterologous constructs, vectors, and transformed plant material herein containing polynucleotides encoding proteins having sweetness regulatory activity. Plant-expressible promoters can be derived from natural plant sources, plant virus sources, and bacteria such as Agrobacterium strains having plant-expressible promoters. Plant-expressible promoters include, among others, the cauliflower mosaic virus promoter (CaMV 35S), the octopine and nopaline synthase promoters (e.g. the nos promoter), the plant ubiquitin promoter (Ubi), the rice actin promoter (Act-1) and the maize Includes alcohol dehydrogenase (Adh-1). Plant-expressible promoters include constitutive promoters, inducible promoters, tissue-specific promoters, developmental stage-specific promoters, and examples of each type of promoter are known in the art. Tissue-specific promoters include promoters that direct expression (eg, the phosphoenolpyruvate promoter (PEP)), particularly in plant roots, plant leaves, fruits, flowers, pollen, or cells involved in active photosynthesis. Developmental stage specific promoters include those that direct expression during fruit ripening, flowering or seed set. Synthetic plant promoters are also known in the art and are useful in heterologous constructs, vectors and transformed plant material (see, eg, Ali S. & Kim W-C (2019) Frontiers in Plant Science, 10, article 1433).

Techniques for regenerating plants from transformed protoplasts, plant cells, callus or other plant tissue are well known in the art and can be used for whole plants and plant parts regenerated from such transformed plant material. Regeneration methods include organogenesis and embryogenesis. Reference: Handbook of plant cell culture. Volume 1: Techniques for propagation and breeding (1983) Edited by D. A. Evans et al., Macmillan (New York); R.H. Smith, Plant Tissue Culture: Techniques and Experiments, 3rd Edition (2012) Academic Press (New York); M.R. See Davey & P. Anthony, Plant Cell Culture: Essential Methods (2010) John Wiley & Sons (New York), especially Chapters 3 and 9.

As a method of introducing the vector into the host, methods commonly used in the art, such as protoplast method and electroporation method, may be used. A transformant of interest can be obtained by selecting a strain into which the vector has been appropriately introduced using indicators such as marker gene expression and/or auxotrophy.

Alternatively, a fragment in which a polynucleotide encoding the Myd polypeptide of the present invention, a regulatory sequence and a marker gene are linked can be introduced directly into the genome of the host. For example, the polynucleotide encoding the Myd polypeptide of the present invention constitutes a DNA fragment in which complementary sequences to the genome of the host are added to both ends of the linked fragment, introduces the fragment into the host, and conducts SOE-PCR on the host genome. It is introduced into the genome of the host by inducing homologous recombination between the DNA fragment and the DNA fragment.

When a polynucleotide encoding the Myd polypeptide of the present invention or a vector containing the same is introduced and the resulting transformant is cultured in an appropriate medium, the MYD cDNA is expressed on the vector, and then the Myd polypeptide of the present invention is produced. A medium used for culturing such a transformant can be appropriately selected by a person skilled in the art according to the type of microorganism of such a transformant.

Alternatively, the Myd polypeptides of the invention can be expressed from polynucleotides encoding the Myd polypeptides of the invention or transcription products thereof using a cell-free translation system. "Cell-free translation system" refers to an in vitro transcription-translation system or an in vitro translation system constructed by adding reagents such as amino acids required for protein translation to a suspension obtained by mechanically disrupting cells of a water column.

The Myd polypeptide of the present invention produced in culture or in a cell-free translation system can be obtained by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography and affinity chromatography alone. Alternatively, they may be separated or purified using a suitable combination. Here, when the gene encoding the Myd polypeptide of the present invention and the secretion signal sequence are operably linked to the vector in the transformant, the resulting Myd polypeptide is more easily removed from the culture because the Myd polypeptide is secreted outside the cell. can be collected appropriately. Myd polypeptides collected from the culture can be further purified by known means.

The present invention also includes a method for producing a protein having sweet taste modulating activity comprising culturing the host cell of the present invention in a medium under conditions that produce a protein having sweet taste modulating activity similar to known sweeteners or compounds.

As used herein, "sweetener", "sweetening compound" or "sweet receptor activating compound" refers to a composition that induces a detectable sweet taste in a subject, such as sucrose, fructose, glucose and other known natural saccharide-based sweeteners. , or known artificial sweeteners such as saccharin, cyclamate, aspartame, etc. discussed further herein, or substances that activate T1R2/T1R3 receptors in vitro. A subject can be a human or an animal.

A sweetener or sweetening composition can be used in an effective amount, which refers to an amount of a sweetening composition of the present invention sufficient to induce a sweet taste in a subject when present in a product for oral administration.

In one embodiment, the instant sweet protein may have sweetness modulating activity. The Myd polypeptides of the present invention may have functional, physical and chemical effects at taste receptors, such as, for example, sweet taste receptors. "Sweetness modulating activity" can refer to inhibition, activation, e.g., agonist or antagonist properties, of a polypeptide of the invention determined using in vitro and in vivo assays for taste delivery. Proteins with inhibitory activity bind stimuli, partially or totally block, reduce, prevent, delay activation, inactivate, desensitize, or down-regulate taste transmission (e.g., antagonists). An activating polypeptide is capable of binding, stimulating, increasing, opening, activating, facilitating, enhancing, sensitizing, or upregulating (e.g., an agonist) activation of a taste signal, e.g., an agonist. can Activated polypeptides are preferred.

Sweetness control also means enhancing taste, such as sweetness, of a particular product for oral administration when administered in combination.

Sweetness modulating activity can be detected in vivo by methods known in the art, such as in vitro methods, or animal or human sensory tests. While not wishing to be bound by any particular theory, Myd is involved in sweet taste activation and is, for example, an agonist of taste 1 receptor 2 (T1R2) and/or taste 1 receptor 3 (T1R3). However, Myd stimulates other taste receptors such as bitter, umami, sour and salty. Such functional effects may be effected by any means known to those skilled in the art, for example spectroscopic properties (eg fluorescence, absorbance, refractive index), hydrodynamics (eg shape), chromatographic or solubility properties, patch clamping, voltage sensitive dyes, whole cell measurement of binding to the taste receptor T1R via changes in current, radioisotope efflux, inducible markers, and transcriptional activation of the T1R gene; ligand binding assay; voltage, membrane potential and conductance changes; ion flux analysis; changes in intracellular secondary messengers such as cAMP, cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; It can be measured by neurotransmitter release and the like.

Sensory assays (human or animal) can also be used to determine whether Myd candidate polypeptides have sweet taste regulating activity. Sensory evaluation is the scientific discipline that analyzes and measures human responses to the composition of food and beverages, such as appearance, touch, smell, texture, temperature, and taste. Sometimes it is necessary to measure using people as tools. Selection of an appropriate method for determining sweetness can be determined by one skilled in the art and includes, for example, a discrimination test or difference test designed to measure the likelihood that two products are perceptibly different. Raters' responses are tallied for accuracy and statistically analyzed to see if any responses are more accurate than expected due to chance alone.

Sensory evaluation is the scientific discipline that analyzes and measures human responses to the composition of foods and beverages, such as appearance, touch, smell, texture, temperature and taste. Sometimes it is necessary to measure using people as tools. The food industry needed to develop this measurement tool because the sensory characteristics of flavor and texture are obvious attributes that cannot be easily measured by instruments. Selection of an appropriate method for determining the organoleptic quality, e.g., sweetness of a protein disclosed herein, can be determined by one skilled in the art, e.g., a discrimination test designed to measure the likelihood that two products differ perceptibly. or a difference test. For sweetness perception, samples and test samples of one or more of, for example, 5% sucrose, 6% sucrose, 7% sucrose, 8% sucrose, 9% sucrose, 10% sucrose were selected from low to high sweetness. It can be ranked by trained panelists in order of sweetness intensity to sweetness. In the context of the present invention, it should be understood that there are many ways by which one skilled in the art can measure sensory differences.

The Brix measurement (or Brix scale) is a well-known application in the food and beverage industry to determine the pure sucrose content of water: 1 degree Brix (°Bx) = 1 g of sucrose/100 g of solution and express the solution as a percentage by mass. 8°Bx is approximately equivalent to an 8% sugar solution. As described in the examples, the purified polypeptide corresponding to SEQ ID NO: 21 was tasted at 0.03 mg/ml (0.2 mL aliquot) by a trained sensory scientist and at 8° Bx (approximately 8% sugar solution). It was found to have a corresponding sweet taste. (See Examples 4, 5, 9 and 10).

In some embodiments, a Myd polypeptide of the invention is at least as sweet as sugar (w/w basis) (eg, 1X), or alternatively 2X less than sugar as measured by any method described above or known in the literature. , 5X, 10X, 50X, 100X, 200X, 400X, 600X, 800X, 1000X, 1500X, 2000X, 3000X, 5000X, 10,000X, 20,000X or more. In another embodiment, the Myd polypeptide is at least 1% (at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%) %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%) as sweet as sugar.

Food, Beverage, Supplements, Medicines

In one embodiment, the present invention includes a composition comprising, consisting essentially of, or consisting of a combination of a product for oral administration and a sweetening composition comprising an isolated Myd polypeptide according to the present invention as described herein. In one embodiment, the combination has an improved sweet taste compared to a product for oral administration lacking the Myd polypeptide (control). In one embodiment, the product for oral administration is not Matyrolomyces terpegioides truffle. The term "consisting essentially of" includes components that are not essential to, and do not materially affect, the function or activity of the product, such as anti-caking agents, fillers, stabilizers (e.g. heat stabilizers), and swelling agents (e.g. eg, maltodextrose, gum acacia, etc.).

In one embodiment, a composition comprising an isolated Myd protein of the invention may include an agent that provides enhanced functionality to the isolated Myd protein. For example, the composition may include a formulation that stabilizes the Myd protein against heat, osmotic pressure, pH or other types of degradation. In one embodiment, the agent stabilizes the Myd protein against thermal degradation. Exemplary compounds for stabilizing the Myd protein include, for example, L-arginine glycine, L-proline, L-histidine, β-alanine, L-serine, L-arginine ethyl ester dihydrochloride, L-argininamide dihydrochloride , 6-aminohexanoic acid, gly-gly, gly-gly-gly, tryptone, betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol, sucrose, hydroxyectoin, trimethyl Amine n-oxide dihydrate, methyl-α-d-glucopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediaminedi Hydrochloride, Guanidine Hydrochloride, Urea, N-Methylurea, N-Ethylurea, N-Methylformamide, Hypotaurine, TCEP Hydrochloride, GSH (L-Glutathione Reduction), Benzamidine Hydrochloride, Disodium Ethylenediaminetetraacetate Dihydrate, magnesium chloride hexahydrate, cadmium chloride hydrate, non-surfactant sulfobetaine 195 (ndsb-195), non-surfactant sulfobetaine 201 (NDSB-201), non-surfactant sulfobetaine 211 (NDSB-211) , non-surfactant sulfobetaine 221 (NDSB-221), non-surfactant sulfobetaine 256 (NDSB-256), taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, taximate pH 7.0 , tetraethylammonium bromide, choline acetate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, thiocyanate Potassium Phosphate, Gadolinium(III) Chloride Hexahydrate, Cesium Chloride, 4-Aminobutyric Acid (GABA), Lithium Nitrate, DL-Malic Acid pH 7.0, Lithium Citrate Tribasic Tetrahydrate, Ammonium Acetate, Sodium Benzenesulfonate, Sodium p-Toluenesulfonate , sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200, polye ethylene glycol monomethyl ether 550, polyethylene glycol monomethyl ether 750, formamide, polyethylene glycol 400, pentaerythritol ethoxylate (15/4 EO/OH), 1,2-propanediol, polyethylene glycol monomethyl ether 1,900, Includes polyethylene glycol 3,350, polyethylene glycol 8,000, polyvinylpyrrolidone k15, polyethylene glycol 20,000, (2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, methyl-β-cyclodextrin do.

In one embodiment, the Myd polypeptide of the sweetening composition is SEQ ID NO:3 (or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17) and an amino acid sequence having at least 80% sequence identity. there is.

In another embodiment, the Myd polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (a) the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID comprising at least one substitution modification to a polypeptide sequence selected from the group consisting of NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is the polypeptide of SEQ ID NO:3 or (b) the polypeptide further comprises a histidine tag, wherein the polypeptide has sweetness modulating activity. For example, a polypeptide comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75 and does not consist of SEQ ID NO:3 don't In certain embodiments, the polypeptide is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

The invention also includes a method of adjusting the taste of a product for oral administration comprising combining the product with an effective amount of an isolated Myd polypeptide as described herein. In one embodiment, the combination has an improved sweet taste compared to a product for oral administration lacking the Myd polypeptide (control). In one embodiment, the product for oral administration is not Matyrolomyces terpegioides truffle.

In one embodiment, the Myd polypeptide of the sweetening composition is SEQ ID NO:3 (or SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17) with at least 80% sequence identity. can

In another embodiment, the Myd polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, has a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (a) the polypeptide is SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide comprises at least one substitution modification compared to a polypeptide sequence selected from the group consisting of SEQ ID NO:3 or (b) the polypeptide further comprises a histidine tag, and the polypeptide has sweetness regulating activity.

Products for oral administration may be food, beverage, dietary supplement compositions or pharmaceutical compositions.

“Products for oral administration” include food, beverage products; It may refer to edible products such as medicinal (pharmaceutical) products, or dietary supplement products such as herbal supplements. As used herein, the term "pharmaceutical" includes both solid and liquid compositions that are ingestible, non-toxic substances that have medical value or contain medicinally active agents such as cough syrups, cough drops, aspirin, and chewable medicinal tablets. . Oral hygiene products are also products for oral administration and include solids and liquids such as toothpaste or mouthwash.

Generally, the present invention provides a food or beverage product with an isolated sweet protein of the present invention in an effective amount, e.g., in an amount up to about 99% by weight relative to the total weight of the food or beverage, e.g., from about 0.01% to about 0.01%. It is contemplated that it may include in an amount of about 99% by weight. All intermediate weights (i.e. 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%,...90%, 95%, 99%) food or beverage products by weight are taken into account, and any intermediate ranges based on these quantities are also considered.

Compositions of the present invention may include solid or liquid media and/or compositions used to prepare a desired dosage form of a Myd polypeptide for administration of the Myd polypeptide in a dispersed/diluted form such that the biological effectiveness of the Myd polypeptide is maximized. As such, a biologically or medically acceptable carrier or excipient" may be included. Edible, biologically or medically acceptable carriers include many common food ingredients, such as water at neutral, acidic or basic pH, fruit or vegetable juices, vinegar, marinades, natural water/fat emulsions such as beer, wine, milk or condensed milk; Edible oils and shortenings, fatty acids, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of these hydrophobic substances in aqueous media, salts such as sodium chloride, flours, solvents such as ethanol, vegetable powders, or solids such as wheat flour. edible diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, tonicity agents; thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like.

Food or beverage products that may be considered in the context of the present invention include baked goods; sweet bakery products (including but not limited to rolls, cakes, pies, pastries and cookies); ready-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings (including but not limited to fruit pie fillings and nut pie fillings such as pecan pie fillings, as well as fillings for cookies, cakes, pastries, confectionery products, etc., such as fat-based cream fillings); desserts, gelatins and puddings; frozen desserts (including but not limited to frozen dairy desserts such as ice cream - including plain ice cream, soft serve and all other types of ice cream - frozen dairy desserts such as non-dairy ice cream, sorbet, etc.); carbonated beverages (including but not limited to soft carbonated beverages); non-carbonated beverages (including but not limited to flavored water and soft non-carbonated beverages such as sweet tea or coffee based beverages); beverage concentrates (including but not limited to liquid concentrates and syrups as well as non-liquid concentrates such as lyophilized and/or powder formulations); yogurt (including, but not limited to, full-fat, low-fat, and fat-free dairy yogurts as well as non-dairy and lactose-free yogurts and frozen equivalents of all of these); snack bars (including but not limited to cereal, nut, seed and/or fruit bars); Bread products (leavened and unleavened bread, yeast and unleavened breads such as soda bread, breads made up of all types of wheat flour, breads made up of all types of secret flours (e.g. potato, rice and rye flour), gluten-free bread); pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spreads (including but not limited to jellies, jams, butter, nut spreads and other spreadable preserves, preserves, etc.); confectionery products (including but not limited to jelly candy, soft candy, hard candy, chocolate, and chewing gum); sweetened breakfast cereals (including but not limited to extruded (kix type) breakfast cereals, flaked breakfast cereals and puffed breakfast cereals); and cereal coating compositions for use in preparing sweetened breakfast cereals. Although not mentioned herein, other types of food and beverage products that typically include one or more nutritive sweeteners are also contemplated in the context of the present invention.

As a result of full or partial replacement of the nutritive sweetener in the food or beverage product of the present invention, the food or beverage product of the present invention can be used in low-calorie or diet products, medical food/products (including pills and tablets), and sports nutrition products, given their solubility. It may be particularly suitable for food or beverage products requiring lower sweetness at the solids level.

In some embodiments, the sweetening compositions of the present invention may be supplemented with other nutritive or non-nutritive sweeteners to form a sweetener system. The sweetener system may include the sweetener composition of the present invention, a bulking agent such as maltodextrose, gum acacia, and the like, and at least one high intensity sweetener. The composition may be provided as a liquid composition or dry blend.

In one embodiment, the invention includes a method of enhancing the sweetness of a product for oral administration comprising the step of adding a Myd polypeptide of the invention.

In another embodiment, the method of the present invention includes a method of improving the sweet taste of an oral product comprising the step of adding a sweetening composition prepared by the method of the present invention to the product for oral administration. The amount added can be determined using methods known in the art, for example sensory tests, as a guide.

The following examples further illustrate the present invention, but of course should not be construed as limiting its scope in any way.

Figure 1 shows the three-dimensional structure of Myd1 predicted based on sequence data using the PHYRE2.0 protein folding prediction tool.
Figure 2 shows a Coomassie-stained SDS-PAGE gel of proteins obtained from fractions of partially purified M. terpegioides gleba.
Figure 3 shows a Coomassie-stained SDS-PAGE gel of the purification step of SEQ ID NO:21 expressed in E. coli. Lane 1: molecular weight standard; lane 3, crude lysate; Lane 4, flow-through fraction of HisPur™ Ni-NTA; lane 5, wash 1; lane 6, wash 2; lane 7, wash 3; lane 8, elution fraction.
Figure 4 shows the concentration-response functions for sweet taste for mycodulcein, aspartame, thaumatin, and rebaudioside A. Data are plotted as the proportion (p) of the response occurring at the 200 mM sucrose-related ("sweet") target. Each data point on the curves for mycodulcein, aspartame, thaumatin, and rebaudioside A was averaged over 32 replicates and averaged over 16 replicates for the sucrose curves. Error bars are SEM. Points for the water and sucrose controls were similarly calculated as averages over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression.
5A shows the predicted tertiary structure of mycodulcein and thaumatin (PDB: 1RQW), monelin (PDB: 2O9U), brazzein (PDB: 1BRZ), and chicken egg white lysozyme (PDB: 1LSN) (protein database). A comparison of the known crystal structures is shown, showing predicted tertiary structural similarity of mycodulcane to other known sweet proteins that contain anti-parallel beta sheets with alpha helices parallel to the beta sheets.
Figure 5B shows the predicted secondary structure of SEQ ID NO:3 superimposed on the putative secondary structure motifs and the location of point mutations within each motif.
Figure 6 shows the mutant his-tagged mycodulcein equalized to equal protein concentrations to each other and his-tagged non-mutant mycodulcein as measured by ELISA for sweetness intensity, sweet perception onset time and sweet perception duration, as measured by ELISA. Shows the result compared to the third.
7A shows SDS-PAGE analysis, Coomassie staining, of fractions eluted from Capto MMC. M: protein marker; Lane 1: eluted fraction, showing low purity after cation exchange. Arrows indicate mycodulcein bands.
7B shows SDS-PAGE analysis, Coomassie staining, two eluted fractions collected during gradient elution from a HiScreen Capto Butyl column analyzed on SDS-PAGE. Lane 1 represents the elution fraction 1 containing no mycodulcein, and lane 2 represents the eluted mycodulcein. The purity of the elution fraction was determined to be -86% by GelAnalyzer. Arrows indicate mycodulcein bands.
Figure 7B shows the protein eluted from the HIC column after SDS-PAGE analysis, Coomassie staining, and chromatography on a HiPrep 26/60 Sephacryl S-200. Lane 1 shows purified his-tag mycodulcein and lane 2 shows purified native mycodulcein. The purity of the elution fraction was determined to be -98% by GelAnalyzer. Arrows indicate mycodulcein bands.

Example

Example 1

Fresh Matyrolomyces terpegioides truffles were obtained on-site from the natural range with appropriate procedures and permits. Fresh samples (29 in total) were purchased from MycoTechnology, Inc. It was shipped to the facility, washed gently with RO water, frozen in liquid nitrogen and stored at -80 °C. The average moisture content of truffles was 83.6% ± 4.6%.

Aqueous extraction of truffles was performed as follows. Eight different truffle samples were ground in liquid nitrogen with a pestle and then 5:1 v/w truffle in 4°C water was added and incubated at 4°C for 30 minutes. The extracted material was then briefly centrifuged at low speed and the filtrate tasted "clean". Sweetness intensity was evaluated from 0 for no sweetness to 10 for very sweet. Sweetness among the samples was evaluated as follows.

Table 1. Sweetness of various truffle samples.

The aqueous extract was stored at 4° C. at pH 7 and pH 2 in sodium phosphate buffer and little or no change in sweetness was observed over 8 days.

Example 2

Purification of the sweet protein Myd from M. terpezoides.

Fresh Matyrolomyces terpegioides truffles were obtained on-site from the natural range using appropriate procedures and permits and stored at -80 °C. A 16.3 g sample was removed from the freezer and ground in liquid nitrogen with a mortar and pestle (white ceramic). Grinding was carried out for 15 minutes to sufficiently grind the tissue to obtain a fine frozen powder. The powder was placed in a 50 mL Falcon tube and 20 mL RO-H ₂ O was added and vortexed to mix the tissue until no ice crystals were visible. The pieces were broken up using a Rotor-Stator set at 20 for 2 X 1 min at 4°C to make a homogeneous solution (H1). The volume of the slurry was adjusted to 53 mL with RO-H ₂ O and centrifuged at 7500 x G for 30 min at 4 °C. The supernatant (S1) from this step was collected in a 2 mL Eppendorf tube and centrifuged at 20,000 x G in a 5417 R and Eppendorf centrifuge at 4 °C for 15 minutes. The pellet (P1) was discarded. Sweetness (via human senses) was detected in the supernatant. The supernatant from this stage was collected and pooled. The supernatant was then filtered through a 0.45 micron syringe filter (cellulose, VWR International), 25 mm and designated S1 + 0.22 um filtration. The filtrate was then washed twice with 38 mL to 50 mL of hexane and the aqueous phase was collected. S1FH stands for S1F + hexane wash. The hexane phase was saved and dried. The aqueous phase was then precipitated with acetone. (At -20°C, 50 mL was added to 33 ml of fraction S1FH and set at -20°C for 30 min.) The sample was centrifuged at 3,000 xg and the precipitate collected. The precipitate was resuspended in 10 mM sodium phosphate pH 6, the supernatant at this stage was referred to as S2 and the precipitate as P2. Supernatant S2 was first applied to an AMICON centrifugal filter device with a molecular weight cutoff of 100 kD to obtain a filtrate (through) portion (referred to as 100F) and a retentate (referred to as 100R); Subsequently, 100F was applied to a device with a molecular weight cutoff of 30 kD to produce a filtrate (30F) and a retentate (30R). The sweet fraction was run through a 100 kD column and held on a 30 kD column (30R) at 100 F. A band was observed at about 13 kDa as indicated by the arrow in SDS-PAGE in FIG. 2, and this band was excised and analyzed for N-terminal sequence by Edman digestion using standard detection methods, such as liquid chromatography and mass spectrometry. was performed to determine the residue for each cycle. A polypeptide of SEQ ID NO:4 was detected.

Example 3 (Identification of RNA)

sample collection

Fresh Matyrolomyces terpegioides truffles were obtained on-site from the natural range with appropriate procedures and permits. A wild isolate (BDP2_18) of Matyrolomyces terpegioides truffle (gleba) was sourced from a natural environment. BDP2_18 was the largest wild truffle removed and the truffle had sweetness characteristics of "sweetness first, more moldy and earthy, low sweet aftertaste".

sample check

Wild isolates from Gleba were shipped frozen to GeneWiz for Internal Transcribed Spacer (ITS) sequencing. The genomic loci sequenced are the ITS 1 & 2 regions. The resulting Sanger sequencing reads were aligned and low quality bases were removed. Then, each sequence was identified through individual BLAST (Basic Local Alignment Search Tool) (Altschul, Gish, Miller, Myers, & Lipman, 1990) search. BLASTn searches were used using nucleotide collections (nr/nt) with unpublished sample sequences excluded. The entry with the highest identity to the wild isolate is Matyrolomyces terpegioides strain rib02.

sample preparation

A wild isolate, BPD2_18, was washed superficially with sterile water, cut into cubes weighing ~ 100 mg, flash frozen in liquid N2 and stored at -80 °C. Delivery was carried out on dry ice for GeneWiz.

RNA sequencing

The following samples were submitted via GeneWiz for Illumina HiSeq, 2x150 bp, single index, ~350M raw paired-end reads (PERs) standard RNASeq per lane. This RNASeq study included polyA+ selection for transcriptome profiling.

GeneWiz extracted RNA using the Qiagen RNeasy Plus Universal mini kit according to the manufacturer's instructions. RNA library preparation was performed using the NEBNext Ultra RNA Library Preparation Kit for Illumina. Illumina adapter sequences are described below.

5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ (SEQ ID NO: 18)

5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7 barcode]ATCTCGTATGCCGTCTTCTGCTTG-3′ (SEQ ID NO: 19)

Fastq sequence files of RNA-seq studies of two strains of Matyrolomyces terpegioides (three replicates each) were trimmed and washed with HTStream to remove the following contaminants. PhiX (a common contaminant in sequencing), rRNA reads, sequencing adapters, low quality and “N” bases, poly-a tracts, primers and reads < 50 bp. Then, rnaSPAdes (Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. rnaSPAdes: a de novo transcriptome assembler and its application to RNA -Seq data. Gigascience. 2019;8(9):giz100.doi:10.1093/gigascience/giz100) was used. Bandage (a Bioinformatics Application for Navigating De novo Assembly Graphs Easily) (Ryan R. Wick, Mark B. Schultz, Justin Zobel, Kathryn E. Holt, Bandage: interactive visualization of de novo genome assemblies, Bioinformatics, Volume 31, Issue 20, 15 October 2015, Pages 3350-3352) performed a tBLASTn search (Gertz EM, Yu YK, Agarwala R, Schaffer AA, Altschul SF. Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST.BMC Biol. 2006;4:41.Published 2006 Dec 7. doi:10.1186/1741-7007-4 -41) was used to visualize and search the assembly for the target peptide.

An RNA transcript was identified, the DNA copy of which has the sequence shown as SEQ ID NO:1, of which SEQ ID NO:2 is the predicted coding sequence based on start and stop codons. A predicted protein with 121 amino acids with an estimated size of 13.3 kD, SEQ ID NO:3 is also provided. Length: 122aa, molecular weight: 13.381 kDa, expected isoelectric point: 8.64, expected charge at pH 7: 1.01.

blast analysis. The identity between the predicted protein SEQ ID NO:3 and the other protein sequences of GENBANK was less than 31%. A hypothetical protein of Pysolidus tintturius (GenBank: KIN98154.1; SEQ ID NO:7) was found to have approximately 31% homology to SEQ ID NO:3. It is called the hypothetical protein M404DRAFT_1005519 [Pysolidus Tintturius Marx 270]. It is hypothesized that SEQ ID NO:7 may also have sweetness regulating activity. A complete cDNA copy of the RNA transcript is SEQ ID NO:5 with the coding sequence given as SEQ ID NO:6.

Example 4 (cloning and heterologous expression of mycodulcein in E. coli; confirmation of sweet taste)

Based on the nucleotide sequence identified as SEQ ID NO:3, three expression vectors were synthesized to express SEQ ID NO:20 containing a histidine tag and three different expression vectors were synthesized by Atum, Inc. (Newark, CA). Cloned into vector backbone: pD454-SR (plasmid pMy_3000), pD454-MR (plasmid pMy_3001), and pD454-WR (plasmid pMy_3002). These are all E. coli IPTG-inducible T7 promoter expression vectors containing the ribosome binding sites of ampicillin-r, lacl, Lac01, Ori_pUC and heavy (M), strong (S) and weak (W) plasmids. E. coli BL21 DE3 (Studier et al. (1986) J. Mol. Biol. 189:113-130) (New England Biolabs) was transformed with pMy_3000, pMy_3001, pMy_3002 using the manufacturer's protocol. In brief, previously frozen competent cells were thawed and mixed with 1 pg -100 ng of plasmid DNA and kept on ice for 30 min. The mixture was subjected to a thermal shock of 42° C. for 45 seconds. Immediately thereafter, the mixture was placed in an ice bath for 10 min. 950 μL of pre-warmed LB medium was added followed by shaking at 225 rpm for 1 hour at 37 °C. For each transfection reaction, cells were diluted 1:10 and 1:100 and plated in 100 μL on antibiotic plates. Growing overnight at 37 °C allowed colonies to fully recover and become prominent. To confirm successful transformation, colony screen PCR (csPCR) was performed to examine the cDNA region of the plasmid, and expression was confirmed by lysate SDS-PAGE. Heterologous expression was confirmed in the E. coli host using shake flask scale directed expression. This process produced strains Z14CE, Z15CE, and Z16CE containing plasmids pMy_3000, pMy_3001 and pMy_3002, respectively. Three strains (Z14CE, Z15CE, Z16CE) were maintained on LB + ampicillin 100 μg/mL agar plates and a negative control (Eco_0001) was maintained on LB agar plates. Incubated overnight for each strain in 50 mL of LB broth in 250 mL culture shake flasks without baffles, shaking at 150 rpm with appropriate antibiotics at 37 °C. Each overnight culture was shaken at 200 rpm in a baffled 1000 mL culture shake flask the following day and seeded in 200 mL of TB broth adjusted to 0.1 OD600 with appropriate antibiotics. When the OD600 reached 0.8, IPTG supplement was added to the medium to a final concentration of 0.66 mM. Shaking was continued at 37 °C for an additional 5 hours. After expression, cells were centrifuged at 4000g for 20 minutes. The supernatant was discarded and the pellet was suspended in about 20 mL of wash buffer (10 mM sodium phosphate buffer pH 7.0). Suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2,000 bar. Disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected and the pellet was discarded. The supernatant containing solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA). Expression was confirmed by running SDS PAGE and observing a 13.1 kD band (Coomassie staining).

Thermo Scientific™ HisPur™ Ni-NTA resin was used to purify the his-tagged protein SEQ ID NO:20 using effective immobilized metal affinity chromatography (IMAC). SEQ ID NO:20 was purified using nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on 6% cross-linked agarose resin. The lysate was loaded onto a prepared IMAC column and equilibrated with binding buffer: 20 mM sodium phosphate monobasic, 0.5 M sodium chloride, 0.1 M imidazole, pH 7.4, and elution buffer: 20 mM sodium phosphate monobasic, 0.5 M sodium chloride, 0.5 M imidazole. was eluted using The column was washed 3 times with binding buffer, then the his-tagged mycodulcein was eluted 4 times with elution buffer, then the eluted fraction was bv ultrafiltered using a 30 kDa MWCO filter followed by a 10 kD filter at 4000x G for 15 min. The filtrate was filtered through. The residue was diluted and spun again for a wash step, repeated three times. Analysis of the purification step by SDS-PAGE is shown in FIG. 3 .

The purified fraction (shown in lane 8 of Figure 3, containing highly purified SEQ ID NO:21) was tasted at 0.03 mg/ml by a trained sensory scientist (0.2 mL aliquot) and 8° Brix ( about 8% sugar solution), which is responsible for the sweetness activity observed in Examples 1 and 2. Sweetness was "clean" sweetness (sugar-like taste), no other flavors, slightly delayed onset and sweet aftertaste.

Example 5 (Pilot Scale Production of Mycodulcein (HIS-TAG))

E. coli strain Z14CE prepared in Example 4 containing the coding sequence SEQ ID NO:20 was tested for its performance during fermentation in a laboratory scale bioreactor. Bioreactor culture was performed in a 10.0 L Bioflo 320 round bottom agitated fermentor (BioFlo/CelliGen 310, New Brunswick Scientific, Edison, NJ, USA). The fermentor was equipped with pH and dissolved oxygen sensors (Mettler Toledo, OH, USA). Temperature was controlled via a stainless steel base filled with water. Agitation was provided by two mounted 6-blade Rushton turbines spaced 47 mm apart with the lowest impeller located just above the bottom of the shaft. Aeration occurred through a perforated pipe sparger ring. Dissolved oxygen (DO) was controlled at 20% of air saturation using a continuous stirring cascade between 500 and 800 rpm and aeration between 5 and 8 L/min with sparged air at high cell density. The pH was adjusted to 7.0 with 5.0 M ammonium hydroxide. Antifoam 204 (Sigma, St Louis, MO, USA) was automatically added to control foaming. The latter was detected using a conductance probe mounted 10 cm above the culture level. The main fermentation medium contained (per liter) 24 g yeast extract, 12 g tryptone, 5.42 mL glycerol, 100 mL phosphate buffer stock (0.17M KH ₂ PO ₄ , 0.72 MK ₂ HPO ₄ ). The medium was adjusted to pH 7.0 with 2M HCl. When the original glucose supply was depleted (signaled by a rise in pH), a feed medium consisting of (per liter) 200 g glucose, 21.1 g (NH ₄ ) ₂ SO ₄ and 19.7 g MgSO ₄ was introduced at an initial flow rate of 1.00 mL/min. Pumped into fermentor. Unless otherwise specified, the initial volume of medium in the vessel was 4.0 L. The inoculum (200 ml) consisted of a culture grown for 16 hours in LB started culture medium in a 1 L baffled shake flask (37° C., 200 rpm). The fermentor temperature was 37°C. After reaching an optical density of 10-20, fermentation was induced with 0.66 mM IPTG and continued for 24 hours thereafter. After a 24 hour induction period, the broth was centrifuged at 4000 g for 20 minutes. The supernatant was discarded and the pellet was suspended in 1 L of wash buffer (10 mM sodium phosphate buffer pH 7.0). Suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated up to 1,500 bar. Disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected and the pellet was discarded. The supernatant containing solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA).

The clarified supernatant prepared in this example was tasted at 0.03 mg/ml by a trained organoleptic scientist (0.2 mL aliquot) and found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). The sweetness was very noticeably sweet, a "clean" sweetness (sugar-like taste) with no other flavors, with a slightly delayed onset and a sweet aftertaste.

The supernatant was stored in aliquots at -20°C and used for further experiments.

Example 6 (Mycodulcein from E. coli ELISA quantification)

A direct ELISA was developed to quantify his-tagged mycodulcein (SEQ ID NO:21) with a horseradish peroxidase (HRP) conjugated antibody to the 6XHis-Tag sequence on the carboxy terminus of SEQ ID NO:21. ELISA can measure mycodulcein concentrations in complex lysates and purified proteins. Recombinant 6XHis-tagged E. coli mycodulcane has a molecular weight of 14.2 kDa and a molar extinction coefficient of 27,960 M ⁻¹ cm ⁻¹ , calculated from the amino acid sequence by methods known in the art. Purity was assessed as > 98% by SDS-PAGE. Mycodulcein protein concentration was determined by absorbance at 280 nm using Beer-Lambert's law and then a standard curve was generated using known concentrations of mycodulcein (μg/ml) for ELISA.

ELISA Procedure: Proteins were bound to the walls of high protein binding 96-well plates in 50 mM carbonate buffer pH 9.4 coating buffer for 30 minutes at room temperature. Plates were washed 3 times with phosphate buffered saline (PBS) pH 7.4 containing 0.02% Tween-20. Non-specific binding sites on the microplate were blocked with 5% BSA in PBS pH 7.4 for 15 minutes at room temperature and washed three times with PBS 0.02% Tween-20. Primary antibodies were diluted (1:1000) in blocking buffer and microplates were incubated for 1 hour and washed 3 times in PBS 0.02% Tween-20. The HRP reaction was run for 8 minutes using 3,3',5,5'-tetramethylbenzidine (TMB) as a colorimetric substrate and stopped with 2N sulfuric acid.

Example 7 (quantitative characterization of the concentration dependence of mycodulcein)

Opertech Bio (Philadelphia, PA) measured the taste properties of purified his-tagged mycodulcein (SEQ ID NO:21) obtained from E. coli as described in Example 5 and purified as described in Example 4. Quantitative characterization of the concentration dependence was performed. The sweetness potency and relative potency of mycodulcein were compared to sucrose and other sweeteners, thaumatin, rebaudioside A and aspartame. The control standard was a solution of 200 mM sucrose, 100 mM NaCl, 0.5 mM quinine and 10 mM citric acid. Figure 4 shows the concentration-response functions for sweet taste for mycodulcein, aspartame, thaumatin, and rebaudioside A. Data are expressed as the percentage (p) of the response occurring at the 200 mM sucrose-related ("sweet") target. Each data point on the curves for mycodulcein, aspartame, thaumatine, and rebaudioside A was averaged over 32 replicates and averaged over 16 replicates for the sucrose curves. Error bars are SEM. Points for the water and sucrose controls were similarly calculated as averages over 128 and 64 replicates, respectively. Curves were fitted by nonlinear regression.

The concentration that induces half of the maximal sweetness response (EC ₅₀ or titer) was derived from non-linear regression. The EC _50s (and 95% confidence intervals) for mycodulcein (MYC), sucrose (SUC), aspartame (ASP), rebaudioside A (REB) and thaumatine (THN) are shown in Table 2. Also provided are equivalents to sucrose on a molar basis and on a weight basis.

Table 2

Evaluation of thaumatin, sucrose and SEQ ID NO:21 was performed using the click all that apply (CATA) time intensity of the sweet sensation of the named analyte in water at 0.045 mg/ml for protein and 10% sucrose. . Methodology: Time Intensity Technique; Data collection software: EyeQuestion, responses recorded every 2.57 seconds; Scaling method: 15-point sweetness scale, eg score 5 = 5% sucrose, score 10 = 10% sucrose; Evaluation protocol: small volume sip, tilt and spit test. Number of panelists: 3-6, number of replicates: 2. All samples were blinded and presented with a random 3-digit code.

TRAINING STRATEGY: Intensive training on the sweetness scale over 6 weeks on a 15-point sweetness scale to confidently perform sweetness assessments. It is necessary to train the time-intensity principle over 3 weeks due to the unique sweet behavior pattern. Samples were tasted with a stopwatch to help record times and reach agreement. Number of panelists: 3-6, number of replicates: 2. All samples were blind and presented with a random 3-digit code. Statistical analysis: Statistical analysis impossible due to the small number of panel members

The maximal strength (Imax) of mycodulcein and thaumatin was approximately 1 point higher on a 15-point scale than sucrose in the amounts tested. Thaumatin and mycodulcein have gentler slopes, resulting in longer peak times and more gradual/longer declines. Sucrose reaches the critical sweetness (intensity <1) sooner than thaumatin and mycodulcein at 162 seconds. When sucrose reaches a threshold level, thaumatine and mycodulcein are perceived as moderate to low strength. Mycodulcein and thaumatin were shown to have similar potency in this experiment, being about 3000 times sweeter than sucrose on a weight basis and about 120,000 times sweeter than sucrose on a molar basis. In these two experiments, mycodulcein was shown to be a high-intensity sweet protein that is 400 to 3000 times sweeter than sucrose by weight.

Example 8 (production and testing of mycodulcein variants for sweetness and heat stability)

A method for identifying potential key residues in mycodulcein. Sweet proteins have no primary sequence identity, but their entire tertiary structure has a sweet finger motif (Tancredi, T., Pastore, A., Salvadori, S., Esposito, V. & Temussi, P. A. Interaction of sweet proteins with their receptor : A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin.European Journal of Biochemistry 271, (2004): 2231-2240.). Sweet proteins have antiparallel beta sheets with alpha helices perpendicular to the beta sheets. The tertiary structures of the sweet proteins thaumatin, monellin, brazzein and lysozyme were analyzed using PyMOL 2.0 (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC) and Phyre2 (Kelley LA et al. The protein Phyre2 web portal for modeling, prediction, and analysis Nature Protocols 10, (2015): 845-858) were compared with the mycodulcein model (Fig. 5A). Twenty-three conservative single point mutations of ionic amino acid residues were generated. The negatively charged glutamine and aspartic acid differ in that they have an extra carbon in the aliphatic chain. Although substituting an arginine with a positively charged lysine is considered a conservative substitution, the guanidinium of arginine can form additional interactions with amino acids, including hydrogen bonds, aromatic and aliphatic contacts. The ionic amino acid mutations were lysine to arginine, arginine to lysine, aspartic acid to glutamic acid, and glutamic acid to aspartic acid. The relative location of each mutation was modeled by PyMOL 2.0 and classified into N-terminus, 3 loop regions, 5 beta-sheets, 1 alpha-helix and C-terminus (see Fig. 5B).

Specifically, the following single mutations were generated and their predicted positions are in Table 3. See also Figure 5B showing an indication of SEQ ID NO:3 predicted secondary structure superimposed on putative secondary structure motifs and the location of Table 3 point mutations within each motif.

Cloning: Eco_0001 aka Escherichia coli BL21 DE3 (E. coli str. B F- ompT gal dcm lon hsdSB(rB-mB-) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS)) (obtained from New England Biolabs# C2527I) was transformed with 23 plasmids (pMy_3018 to pMy_3040) using the manufacturer's protocol. Briefly, previously frozen competent cells were thawed and mixed with 1 pg - 100 ng of plasmid DNA and kept on ice for 30 minutes. The mixture was subjected to a thermal shock of 42° C. for 45 seconds. Immediately thereafter, the mixture was placed in an ice bath for 10 min. 950 μL of pre-warmed LB medium was added followed by shaking at 225 rpm for 1 hour at 37°C. For each transfection reaction, cells were diluted 1:10 and 1:100 and plated in 100 μL on antibiotic plates. Growing overnight at 37°C allowed colonies to fully recover and become visible. This process produced strains Z18CE to Z40CE containing plasmids pMy_3018 to pMy_3040 sequentially. Heterologous expression was confirmed in the E. coli host using shake flask scale directed expression. After transformation, mutants were plated and maintained on LB + Ampicillin 100 μg/mL agar plates.

Plates were incubated at 37° C. for 16 hours. Colony screen PCR was performed to confirm genotype using colony screening primers designed to interrogate the flanking region along with the cDNA region of the plasmid. Successful transformation produces DNA fragments of a certain size, whereas no PCR bands appear in the absence of negative and template controls. Successful transformation was observed for all mutants. Heterologous expression was confirmed in the E. coli host using shake flask scale directed expression.

Twenty-three strains (Z38CE to Z60CE) were maintained on LB + Ampicillin 100 μg/mL agar plates and a negative control (Eco_0001) was maintained on LB agar plates. Incubated overnight for each strain in 50 mL of LB broth in 250 mL culture shake flasks without baffles, shaking at 150 rpm with appropriate antibiotics at 37 °C. Each overnight culture was shaken at 200 rpm in a baffled 1000 mL culture shake flask the next day and seeded in 200 mL of TB broth adjusted to 0.1 OD600 with appropriate antibiotics. When the OD600 reached 0.8, IPTG supplement was added to the medium to a final concentration of 0.66 mM. Shaking was continued at 37° C. for an additional 5 hours. Cells were then collected by centrifugation at 5000 g at 4° C. for 5 minutes. E. coli cells were washed once with cold dH ₂ O and again centrifuged at 5000 g for 10 min at 4 °C. To confirm successful expression, cell lysates were generated using liquid nitrogen and a mortar and pestle. The cell pellet was resuspended in 10 mL of cold dH ₂ O and the crude lysate was spun at 20,000 g for 5 min at 4 °C. Finally, the supernatant was aspirated and filtered through a 0.2 μm PES filter and subjected to SDS-PAGE protein electrophoresis. Crude lysate was tasted to identify sweet tasting mutants. Table 3 shows the test results.

Table 3. Sweetness test results for single point mutations in mycodulcein.

All 16 of the sweet-tasting variants (Z38CE, Z39CE, Z41CE, Z45CE, Z47CE, Z48CE, Z49CE, Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, Z60CE) were added using 200 mL of medium. reappeared. After expression, cells were centrifuged at 4000g for 20 minutes after a 24 hour induction period. The supernatant was discarded and the pellet was suspended in approximately 20 mL of wash buffer (10 mM sodium phosphate buffer pH 7.0). Suspended cells were disrupted in a high-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a maximum of 2,000 bar. Disrupted cells were centrifuged at 13,000 g (30 min), the supernatant was collected and the pellet was discarded. The supernatant containing solubilized protein was filtered through a 0.22 μm PES membrane unit (Millipore, Burlington, MA, USA). Material was then isolated via IMAC purification as described in Example 4.

A trained sensory scientist tasted the purified samples. Mycodulcein stocks were diluted to equal protein concentrations as determined by ELISA. Subjects followed an Institutional Review Board approved rinse and spit protocol. 0.2ml of each purified mutant strain was placed on the tongue, and the sweetness perception intensity, sweetness perception onset time, and sweetness perception duration were recorded. Results are shown in Figure 6 and described below.

Effect of Conservative Point Mutations on Mycodulcein Sweetness

To correlate the effect of conservative single point mutations on sweet taste, published mutations of thaumatin, brazzein, monelin and lysozyme were compared to mycordulcein. Since the goal was to match the mycodulcein time and intensity profile to sucrose, sensory analysis was used to determine sucrose equivalence, onset and total duration. Sucrose has fast onset, high intensity and fast duration. Thus, mutations that decrease expression and duration are desirable, as are mutations that match or enhance sucrose equivalence. Mutations that increase expression and overall duration are just as undesirable as those that decrease sucrose equivalence. See Figures 5B and 6.

N-Terminus-External- D3E

Conservative changes of a single mutation of D3E at the outer N-terminus resulted in loss of sucrose equivalence and duration; However, expression decreased only slightly. These results suggest that the charge, size and polarity of the sweet protein N-terminus are important for sweet taste and protein stability.

Beta Seat 1 - External - K11R

Conservative changes of a single mutation in K11R resulted in a slight increase in sucrose equivalency and a moderate decrease in expression; However, the duration of sweetness increased dramatically. These results suggest that K11 is an important residue for binding to the sweet receptor T1R2/T1R3 and may affect the off rate of mycodulcein at the receptor.

Region between beta sheets 2 and 3 - outside - K26R - linker region

A conservative mutation in K26 resulted in a slight decrease in sucrose equivalence, indicating that this conservative mutation does not significantly affect protein functionality.

Loop 2 Area - Outside - K51R

Molecular modeling showed that all putative loop region mutations were solvent exposed. Except for a modest decrease in onset, only a slight decrease in sucrose equivalence and total duration was observed in sensory studies, indicating that these conservative mutations do not significantly affect protein functionality.

Loop 2 Area - Outside - R57K

Mutant R57K has a significant inhibitory effect on expression, sucrose equivalence and total duration.

Beta Seat 4 - External - R66K

Mutant R66K significantly reduced sucrose equivalence and total duration, while slightly increased expression. The R66K mutation may be a key residue in affinity and off-rate for sweet receptors.

Loop 3 Area - Outside - D69E

The mutation at D69E results in sucrose equivalency, a slight decrease in duration and a slight increase in duration. At this site, this region is expected to be relatively insensitive to conservative mutations.

Alpha-helical region - outside - D85E and D86E

Both D85E and D86E have similar effects on mycodulcein sensory properties. Reduced sucrose equivalence and duration for D85E and D86E. Expression was similar to control.

Outside the C-terminus - D97E, K103R, R106K and E117D had minimal effects compared to controls, suggesting that these regions are relatively insensitive to conservative mutations. However, R110K and K120R showed a decrease in sugar content and duration, but the expression was similar.

As shown in Table 3, conservative single point mutations in R20K, E35D, K44R, D46E, D52E, R75K and D94E resulted in loss of protein expression. These data suggest that these residues may be involved in protein folding or expression within the E. coli host. The predicted tertiary structure model discussed above in this example supports protein folding errors due to these changes since all these residues are included in the predicted beta-sheet.

references

Korz, D. J., Rinas, U., Hellmuth, K., Sanders, E. A. & Deckwer, W.-D. Simple fed-batch technique for high cell density cultivation of Escherichia coli. Journal of Biotechnology 39, 59-65 (1995).

Norsyahida, A., Rahmah, N. & Ahmad, RMY Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli. Letters in Applied Microbiology 49, 544-550 (2009).

Example 9.

IMAC of 16 sweet mutants as described in Example 8, where thermal stability was normalized to 0.04 mg/mL using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Inc. Fremont, CA) according to instructions provided by the manufacturer. This was done on protein samples from purification. That is, the individual reaction to measure the thermal change relies on mixing: mycodulcein, 36 μg/ml in 25 mM sodium phosphate buffer (pH 7.4) with the reagents provided in the kit according to the manufacturer's instructions. For thermal shift measurements, Bio-Rad CFX96 Touch system using BR clear plate (scan mode SYBER/FAM only, 25°C for 30 seconds, melting curve 25°C to 95°C, 0.5°C increment for 10 seconds plus plate lead) was used. Tm is Schulz, MN, Landstrφm, J. & Hubbard, RE MTSA—A Matlab program to fit thermal shift data. It is determined based on the midpoint determined for the curve fitted to the experimental data with a 5-parameter equation using a technique described in Analytical Biochemistry 433, 43-47 (2013). The results (Table 4) show that thermal stability is minimally affected by amino acid changes in the tested mutants.

Table 4. Thermal stability test results for mutations.

Example 10 (cloning and heterologous expression of mycodulcein in Saccharomyces cerevisiae; confirmation of sweet taste)

Based on the nucleotide sequence identified as SEQ ID NO:3, two expression vectors expressing SEQ ID NO:21 (pMy_4003) (his-tagged) and SEQ ID NO:3 (pMy_4002) (native) mycodulcein were prepared. Synthesized and cloned by Atum, Inc. (Newark, Calif.) into the Atum vector non-secreted backbone pD1234 containing the URA3 marker and the strong constitutive promoter GPD. The transformation procedure involves creating electrocompetent cells and then introducing the expression vector through electroporation. Briefly, electrocompetent cells are generated by first growing the cells between early and mid-log phases by washing several times to remove salts from the growth medium. After mixing 1-5 μg of the expression vector, the samples are set up on a Gene Pulser II electroporator as follows (charging voltage: 1.5 kV, capacitance: 25 μF, resistance: 200 Ω). Immediately add 1 mL of pre-heated 30 °C YPD and shake the suspension at 200-250 rpm for 1-2 h at 30 °C. After transformation, mutants were plated and maintained on SC-ura agar plates. This process produced strains Z19ES and Z20ES containing plasmids pMy_4002 and pMy_4003, respectively. Colony screen PCR (csPCR) was performed to examine the cDNA region of the plasmid. Thus, successful transformation yielded a DNA fragment of 303 bp, whereas no PCR bands and expression were confirmed in the absence of negative and template controls.

Two strains (Z19ES, Z20ES) were maintained on SC-ura agar plates, while negative controls were maintained on SC agar plates. Overnight cultures for each strain were grown overnight at 150 rpm at 37°C in 50 mL of SC-ura/SC broth in 250 mL culture shake flasks without baffles. Each overnight culture was inoculated into 200 mL of SC-ura/SC (O-RDL-R10_TB medium) broth in a baffled 1000 mL culture shake flask, shaken at 200 rpm at 30 °C and adjusted to 0.02 OD600. Agitation was continued at 30° C. for an additional 48 hours. Cells were then collected by centrifugation at 5000 g at 4° C. for 5 minutes. Thereafter, the S. cerevisiae cells were washed with cold dH ₂ O and centrifuged again at 5000g at 4° C. for 5 minutes. Cells were lysed using liquid nitrogen and a barracks bowl to confirm successful expression. The cell pellet was resuspended in 10 mL of cold dH ₂ O and the lysate spun at 20,000g for 5 min at 4°C. The supernatant was filtered through a 0.2 μm PES filter. The filtrate (both strains) was found to be sweet by the method described in Example 3.

Thermo Scientific™ HisPur™ Ni-NTA resin was used to purify the his-tagged protein SEQ ID NO:20 from S. cerevisiae using effective immobilized metal affinity chromatography (IMAC). SEQ ID NO:20 was purified using nickel-charged nitrilotriacetic acid (NTA) chelate immobilized on 6% cross-linked agarose resin. The lysate was loaded onto the prepared IMAC column, the column was washed 3 times with 0.02 M imidazole in PBS, then the his-tagged mycodulcein was eluted 4 times with 0.3 M imidazole in PBS, and the 50 kDa MWCO filter was The elution fraction was ultrafiltered using a 3 kDa MWCO filter and then concentrated and desalted.

Example 11 (Purification of Natural Mycodulcein from Strain Z19ES (S. cerevisiae))

Three chromatographic techniques were evaluated for their effectiveness for the purification of native mycodulcein (SEQ ID NO:3) expressed in S. cerevisiae: cation exchange (CIEX), hydrophobic interaction (HIC) and size exclusion. Chromatography (SEC).

Cation exchange evaluation.

The isoelectric point for native mycodulcein was determined to be -9.5 by isoelectric focusing, suggesting that the cation exchange column could be successful in purifying mycodulcein. Clarified cell lysates prepared as described in Example 10 were mixed with 2x starting buffer to obtain cell lysates in 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0, and stored at 4°C for future use. The purification procedure was performed on an AKTA Explorer 100 system (GE Healthcare, Sweden) and eluted proteins were monitored at 280 nm and 215 nm on a UV detector UV-900 (GE Healthcare, Sweden). The binding conditions of native mycodulcein were screened using PreDictor plates (GE Healthcare, Sweden) pre-filled with CIEX resin. The pre-filled plates contain three main resins: Capto S (strong CIEX), Capto MMC (weak CIEX) and SP Sepharose Fast Flow (strong CIEX). The lysates were dialyzed against 20 mM sodium phosphate dibasic and screened for various pH values ranging from 4 to 9, then the optimal conditions were scaled up using a HiScreen column. Equilibration was performed using 25 mM sodium phosphate dibasic at pH 5 at a flow rate of 3 mL/min. Bound proteins were eluted by an increasing sodium chloride gradient from 0 to 1 M using 25 mM sodium phosphate dibasic, 1 M sodium chloride at pH 7. Different binding and elution conditions were screened under which the weak cation exchanger Capto MMC exhibited the best binding capacity at pH 5. However, due to the low purity (25%) after this step, an alternative purification step was sought. Figure 7A shows the PAGE analysis of the fractions eluted from Capto MMC. M: protein marker; Lane 1: eluted fraction, showing low purity after cation exchange. Arrows indicate mycodulcein bands.

HIC evaluation.

Cell lysates were also subjected to hydrophobic interaction chromatography (HIC) using a HiScreen CaptoButyl column (Cytiva, Sweden); Equilibration was performed using 5 column volumes of 50 mM sodium phosphate, 1 M ammonium sulfate at pH 7.0. The cell lysate was then loaded onto the column at a flow rate of 3 mL/min. Elution of bound proteins was performed by a decreasing ammonium sulfate gradient from 1 to 0 M using 50 mM sodium phosphate at pH 7.0. All fractions obtained were analyzed by SDS-PAGE.

Figure 7B shows the two elution fractions collected during gradient elution from the HiScreen Capto Butyl column analyzed on SDS-PAGE. Lane 1 represents the elution fraction 1 containing no mycodulcein, and lane 2 represents the eluted mycodulcein. The purity of the elution fraction was determined to be -86% by GelAnalyzer.

SEC evaluation.

The eluted fraction containing native mycodulcein was then further purified using size exclusion chromatography (SEC) HiPrep 26/60 Sephacryl S-200 HR column (Cytiva, Sweden), 50 mM sodium phosphate and 150 mM sodium phosphate at pH 7.0. Elution was performed with a buffer containing NaCl. Fractions were collected, then concentrated, desalted using a 3 kDa molecular weight cutoff (MWCO) centrifugal filter (Millipore-Sigma, Germany) and analyzed by SDS-PAGE.

Summary: Although native mycodulcein binds successfully to the weak cation exchanger Capto MMC, the relatively low purity of the elution fraction makes CIEX a less desirable capture/intermediate purification step. On the other hand, higher purity fractions were obtained from HIC and Capto Butyl columns. According to the SDS-PAGE analysis, the impurity had a relatively high molecular weight, which made SEC titration a candidate for obtaining high-purity natural mycodulcein.

The purity after HIC/SEC is about 98% by GelAnalyzer on SDS-PAGE. 7C shows the protein eluted from the HIC column after chromatography on HiPrep 26/60 Sephacryl S-200. Lane 1 shows purified his-tag mycodulcein and lane 2 shows purified native mycodulcein.

The purified natural protein from S. cerevisiae was tasted at 0.03 mg/ml by a trained sensory scientist (0.2 mL aliquot) and was found to have a sweetness equivalent to 8° Brix (approximately 8% sugar solution). Turns out. The sweetness was very noticeably sweet, a "clean" sweetness (sugar-like taste) with no other flavors, with a slightly delayed onset and a sweet aftertaste.

Example 12 (application data)

His-tagged mycodulcein prepared as in Example 5 and purified as described in Example 5 was tested in a yogurt base. The yogurt base had the following recipe (Table 5):

Table 5.

Cane sugar is added as a carbon source for yogurt culture and is at least partially consumed by the culture. Mycodulcein was added to approximate the sweetness of 8° to 10° Brix of sugar, resulting in a final concentration of 0.05 mg/ml in yogurt base. Taste tests were conducted by experienced sensory scientists and the sweetness of yogurt was found to correspond to 8° Brix (approximately 8% sugar solution). The sweetness was very noticeably sweet, a "clean" sweetness (sugar-like taste) with no other flavors, with a slightly delayed onset and a sweet aftertaste.

His-tagged mycodulcein prepared as in Example 5 and purified as described in Example 5 was tested in whole milk; Nondairy Pea Based Milk (Water, 93.75%, Pea Protein, 4.2%, Canola Oil, 1.7%, TIC Gum Blend Pro 181 AG (Acacia + Gellan) 0.3%, Sunflower Lecithin 0.05%); cold coffee; and water (control) at a final concentration of 0.04 mg/ml, expected to provide a sweetness level between 8° and 10° Brix of sugar. The taste test confirmed that the sweet protein delivered a sweetness level of 8° to 10° Brix in all samples and all samples had similar sweetness intensity, onset and duration to the water control.

All references, including publications, patent applications and patents cited herein, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated for incorporation by reference in its entirety.

The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (particularly in the context of the following claims) should be interpreted as follows. Include both the singular and plural unless otherwise specified herein or otherwise clearly contradicted by context. The use of a list of one or more items (eg, "at least one of A and B") following the term "at least one" shall be construed to mean one item selected from among the listed items (A or B). ) or a combination of two or more of the listed items (A and B). The terms "comprising", "having", "including" and "including" are to be interpreted as open-ended terms (ie, meaning "including but not limited to") unless otherwise specified. The term "consisting of" is to be construed as a closed term (ie, excluding elements or steps other than those listed). The term "consisting essentially of" permits the inclusion of components or steps that are not essential to, and do not materially affect, the function or activity of a product or method.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range unless otherwise indicated herein, and each separate value is stated as if it were individually recited. included in here. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples or use of exemplary language (eg, “such as”) provided herein is merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of all possible variations of the foregoing elements is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

SEQUENCE LISTING <110> MycoTechnology, Inc. <120> SWEET PROTEIN FROM TRUFFLE <130> 3388820: 0640.30A WO <150> US 63/044245 <151> 2020-06-25 <160> 75 <170> PatentIn version 3.5 <210> 1 <211> 1072 <212 > DNA <213> Mattirolomyces terfezioides <400> 1 caagtcttta agtgttgatc gagcagtaga aatattaatc gcccaattaa ttgctcttac 60 tctaggaaat gtagtactca agttggccct ctaagtccct gattcagtgt cactttttct 120 cctttgacag taggctagat tctagctctt gtgtccgttt cttaaaatcc tgatatggca 180 ccttagcgtc agagcagaat acctgttctc atacaccttg gctatgatcc gatatttaaa 240 tagagcaatc acccttctgg atatcttccc catagctccc attgacctca aacattttct 300 aatcccctat accagccata ttacttgtct aagcacttca cattcttcct cacaatacga 360 aacctcctaa aatgcctgat ctctcctcct tcattacgat taagaacaac tctaaccacg 420 tcttcactcg gacggcaatt tattctaagt atgccgctgt gcagtggagt cccgagccgc 480 aactctcaat ctctcccggc aaatgggatt tgtttatttt aaaggacatc ttgtctatcc 540 gtgggacttc gggatatgta caatatcggg ttggggatgg tcctggatgg gttagggtca 600 ccttttcttc tctagtcggg gccgatgaag tggcagagtg gagctcaggt gacctacctg 660 atggctttgt tctccaaaaa ccagtt cgca ctgggtccag gcctttgcag gcaacgttcg 720 aggctacaaa acagtaaaga tcgatgatgc ctaatatgcc tccataacac tgacccgcgt 780 gcacatggcc gcatgaatga taagggggat atcgatgatg atgggattag ttattggaat 840 tttcacaatg gacgtcggct tggatttaca ataatcgttt catttgtatt caaatattcc 900 tatttccttg ggtttttgta tttatctcct tcatcacgcc ttctgaggcc gtgggaagat 960 gaatatgtaa tcaaaagaag ttaggatatg catcatgtac agaaagtgga ccgcaacccc 1020 ttcagcgaaa tgttataaag atgatatcta agacgccaaa gcacattctc ag 1072 <210> 2 <211> 366 <212> DNA <213> Mattirolomyces terfezioides <400> 2 atgcctgatc tctcctcctt cattacgatt aagaacaact ctaaccacgt cttcactcgg 60 acggcaattt attctaagta tgccgctgtg cagtggagtc ccgagccgca actctcaatc 120 tctcccggca aatgggattt gtttatttta aaggacatct tgtctatccg tgggacttcg 180 ggatatgtac aatatcgggt tggggatggt cctggatggg ttagggtcac cttttcttct 240 ctagtcgggg ccgatgaagt ggcagagtgg agctcaggtg acctacctga tggctttgtt 300 ctccaaaaac cagttcgcac tgggtccagg cctttgcagg caacgttcga ggctacaaaa 360 cagtaa 366 <210> 3 <211> 121 <212> PRT <213> Mattirolom yces terfezioides <400> 3 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 4 <211> 20 <212> PRT <213> Mattirolomyces terfezioides <400> 4 Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His Val 1 5 10 15 Phe Thr Arg Thr 20 <210 > 5 <211> 873 <212> DNA <213> Pisolithus tincturius <400> 5 tcgtccgagc agataaacat cgacgggcag gatgtcattt ctactgaatg tctggaacgc 60 tgtccaggcc cgaggtacaa tcttcctatc aacgacgc ag acgaggtaca atactgctgt 120 caatgctaca aaatcatgcc gggtcctgtg catggccgtc tgaggaggtt cttctggtag 180 aagtttaaaa ggtctctggg cgacgggtgt aacttgccaa tttcccctac atccgaacat 240 ggcagatgaa cagaaggaac tcgcaaccaa cgtagccgac catggtctta ctggccaact 300 tccctcatca aacgaaacca cggagggact cggagtcacg caagactgct cttgctacat 360 cacgctccac aatgggaccg accatgagct tgtgctcgtc tacgctcaag agaaacacgg 420 tgaatggaag acccgccctg cggaaaccgt gagccagaag agcaatatca agttttggct 480 caaagactta ttccttggtc ctggagcaga gggtatggtg aagtatcgaa tcggaagtac 540 cgagcacaag gtgcagatga acttcagctg tcctatgtct tctcccaact cggcgtcctg 600 gagtcaaggt gaacatgaga ttccaggcat ctggttgccc tgtccggaat acaataaatc 660 tgatgcgttg catgccgtgt ttgaagtaca acctgggaat taaggcgtcg gggcggggag 720 acgtactata ccccattgtc gatagcctct ggaagtgtca tcataatgac tgtttgtttt 780 gtcattgacc ggcgacttgt cattttgagt tcgtttccct tggcctgagg cgcacttacc 840 gggcatcgtc atagagctac tgcttaccac aaa 873 <210> 6 <211> 464 <212> DNA <213> Pisolithus tincturius <400> 6 atggcagatg aacagaagga actc gcaacc aacgtagccg accatggtct tactggccaa 60 cttccctcat caaacgaaac cacggaggga ctcggagtca cgcaagactg ctcttgctac 120 atcacgctcc acaatgggac cgaccatgag cttgtgctcg tctacgctca agagaaacac 180 ggtgaatgga agacccgccc tgcggaaacc gtgagccaga agagcaatat caagttttgg 240 ctcaaagact tattccttgg tcctggagca gagggtatgg tgaagtatcg aatcggaagt 300 accgagcaca aggtgcagat gaacttcagc tgtcctatgt cttctcccaa ctcggcgtcc 360 tggagtcaag gtgaacatga gattccaggc atctggttgc cctgtccgga atacaataaa 420 tctgatgcgt tgcatgccgt gtttgaagta caacctggga atta 464 <210> 7 <211> 154 <212> PRT <213> Pisolithus tincturius <400> 7 Met Ala Asp Glu Gln Lys Glu Leu Ala Thr Asn Val Ala Asp His Gly 1 5 10 15 Leu Thr Gly Gln Leu Pro Ser Ser Asn Glu Thr Thr Glu Gly Leu Gly 20 25 30 Val Thr Gln Asp Cys Ser Cys Tyr Ile Thr Leu His Asn Gly Thr Asp 35 40 45 His Glu Leu Val Leu Val Tyr Ala Gln Glu Lys His Gly Glu Trp Lys 50 55 60 Thr Arg Pro Ala Glu Thr Val Ser Gln Lys Ser Asn Ile Lys Phe Trp 65 70 75 80 Leu Lys Asp Leu Phe Leu Gly Pro Gly A la Glu Gly Met Val Lys Tyr 85 90 95 Arg Ile Gly Ser Thr Glu His Lys Val Gln Met Asn Phe Ser Cys Pro 100 105 110 Met Ser Ser Pro Asn Ser Ala Ser Trp Ser Gln Gly Glu His Glu Ile 115 120 125 Pro Gly Ile Trp Leu Pro Cys Pro Glu Tyr Asn Lys Ser Asp Ala Leu 130 135 140 His Ala Val Phe Glu Val Gln Pro Gly Asn 145 150 <210> 8 <211> 121 <212> PRT <213> Artificial Sequence <220> < 223> Mutated protein from Mattirolomyces terfezioides <400> 8 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys His Asn Ser His His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Gln Glu Pro His Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Ala Ala Gln Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Trp His Gln Gln Val Thr Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <220 > <223> Mutated protein from Mattirolomyces terfezioides <400> 9 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Thr Asp His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Gln Pro Gln Leu Ser Ile Ser His Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly His Gly Pro Asp Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Trp His Glu His Val Pro Met Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 10 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 10 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Lys Pro Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Ser Glu Ser Gln Leu Ser Ile Pro Ser Gly Glu Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Val Gly Ser Cys Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Ser Val Gln Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 11 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 11 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn His Thr Asn Arg 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Thr Pro Glu Ser Leu Met Ser Ile Ser Pro Gly Thr Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Cys Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Gln Met Gly Thr Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 12 < 211> 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 12 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asp Asn Ser Asn Gln 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Cys Pro Glu Leu Pro Arg Ser Ile Ser Leu Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Ala Leu Ala Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Leu Val Arg Asn Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 13 <211> 121 <212> PRT <213> Artificial Sequence <220> < 223> Mutated protein from Mattirolomyces terezoides <400> 13 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys His Asn Ala His His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Gln Glu Gln Gln Leu Ser Ile Ala Pro Gly Thr Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Arg Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Gln Val Arg Lys Arg Ala Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 14 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 14 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn His Ser Asn Asn 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Pro Arg Gly Pro Leu Leu Ser Ile Ser Pro Arg Gln Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Arg Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Arg Val Gly Asn Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 15 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser His Gln Pro Arg Pro Ser Ile Trp Pro Asp Met Trp A sp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Asp Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Arg Met Pro Asp Pro Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 16 <211 > 121 <212> PRT <213> Artificial Sequence <220> <223> Mutated protein from Mattirolomyces terfezioides <400> 16 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asp Asp Pro Asn Gln 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu His Gln Phe Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Cys Asp Cys Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly P he Val Phe Gln Thr Pro Val Thr Thr Gly Pro Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <223 > Mutated protein from Mattirolomyces terfezioides <400> 17 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Lys Thr Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Thr Gln Gln Pro Gln Ile Ser Ile Thr Gln Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Asp Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Arg Lys Pro Leu Arg Met Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln 115 120 <210> 18 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Adapter Sequence <400> 18 aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58 <210> 19 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Adapter Sequence <220> <221> misc_feature <222> (34) ..(34) <223> insertion of i7 barcode <400> 19 gatcggaaga gcacacgtct gaactccagt cacnatctcg tatgccgtct tctgcttg 58 <210> 20 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Modified coding sequence for E. coli expression for protein from Mattirolomyces terfezioides with His-Tag <220> <221> CDS <222> (1)..(384) <400> 20 atg ccg gat ttg agc tcg ttc att act att aag aat aac tct aac cat 48 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 gtt ttt acg cgt acc gca atc tat tcc aaa tat gcc gca gtc cag tgg 96 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 agc ccg gaa ccg cag ctg agc att agc cca ggt aaa tgg gac ctg ttc 144 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 atc ctg aaa gac atc ctg agc atc cgc ggt acg tcc ggc tac gtg cag 192 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 tac cgt gtc ggc gac ggt ccg ggc tgg gtg cgt gtt acc ttt agc agc 240 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser Ser 65 70 75 80 ctg gtg ggt gcg gac gaa gtt gct gag tgg agc agc ggt gat ctg ccg 288 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 gat ggc ttt gtt ctg caa aag cct gtc cgc acc ggt agc cgt ccg ctg 336 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 caa gcg acg ttc gag gcg acc aag caa cat cac cat cac cac cac taa 384 Gln Ala Thr Phe Glu Ala Thr Lys Gln His His His His His His 115 120 125 <210> 21 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln His His His His His His 115 120 125 <210> 22 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Modified coding sequence for Saccharomyces cerevisiae expression for protein from Mattirolomyces terfezioides with His-Tag <400> 22 atgcctgatt tatcaagttt tatcaccatt aaaaataact ctaaccatgt ttttactaga 60 acagccatct actcaaagta cgcagccgtc caatggtccc cagaacctca gttgtctata 120 tctccaggta aatgggatct tttcatctta aaagatattc taagtattag aggcacttct 180 ggttacgtac agtatcgtgt tggtgatgga cctggttggg ttagagtaac attcagctca 240 ttggttgggg ctga cgaagt ggctgagtgg tcctcaggtg acctcccaga tggcttcgtg 300 ctgcaaaagc cagtcagaac tggatctaga ccattgcaag cgacattcga agcaacaaag 360 caatga 366 <210> 23 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence for modfied protein from Mattirolomyces terfezioides including His-Tag <400> 23 atgccggaat tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 24 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His- Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 24 Met Pro Glu Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210 > 25 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 25 atgccggatt tgagctcgtt cattactatt agaaataact ctaaccatgt ttttacgcgt 60 accgcaatct cagctgagtagccat 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacg ttcga ggcgaccaag 360 caacatcacc accacacacca ctaa 384 <210> 26 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absnet or Histidine <400> 26 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Arg Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 27 <211> 384 <212> DNA <213> Artificial Sequence <220> < 223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 27 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgaaa 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 28 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 28 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Lys Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Il e Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 29 < 211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 29 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attcccgcta tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgac caag 360 caacatcacc accaccacca ctaa 384 <210> 30 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> ( 122)..(127) <223> Absent or Histidine <400> 30 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Arg Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 31 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 31 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggatccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 32 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides <221 with optional2> His-Tageoides <2> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 32 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Asp Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys As p Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 33 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 33 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggtc gctgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caa catcacc accaccacca ctaa 384 <210> 34 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122) ..(127) <223> Absent or Histidine <400> 34 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Arg Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 35 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 35 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggaact gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 36 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature < 222> (122)..(127) <223> Absent or Histidine <400> 36 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Glu Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Se r Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 37 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 37 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg cgcgacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga 3 ggcgaccaag ccacca ctaa 384 <210> 38 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122). .(127) <223> Absent or Histidine <400> 38 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Arg Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 39 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified pro tein from Mattirolomyces terfezioides with His-Tag <400> 39 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagaaatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 40 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222 > (122)..(127) <223> Absent or Histidine <400> 40 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Glu Ile Leu Ser Ile Arg Gl y Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 41 <211> 384 <212> DNA <213 > Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 41 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatcaa aggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg ctaa 384 <210> 42 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..( 127) <223> Absent or Histidine <400> 42 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Lys Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 43 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Ma ttirolomyces terfezioides with His-Tag <400> 43 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtacaaagt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 44 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> ( 122)..(127) <223> Absent or Histidine <400> 44 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gl y Tyr Val Gln 50 55 60 Tyr Lys Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 45 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 45 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgagggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 46 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Matirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122). .(127) <223> Absent or Histidine <400> 46 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Glu Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 47 <211> 384 <212> DNA <213 > Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 47 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt cc gggctggg tgaaagttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 48 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 48 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Lys Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 49 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 49 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggaagaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 50 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Matirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127 ) <223> Absent or Histidine <400> 50 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Glu Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 51 <211> 384 <212> DNA <213> Artificial Seguence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 51 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgc gtgttac ctttagcagc 240 ctggtgggtg cggacgatgt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 52 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 52 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Asp Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 53 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified from Mattirolomyces terfezioides with His-Tag <400> 53 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgattgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 54 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223 > Absent or Histidine <400> 54 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Ph e Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Asp Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 55 <211> 384 <212> DNA <213> Artificial Sequence < 220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 55 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac cttt agcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg agctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 56 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 56 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Glu Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 57 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 57 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga aggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 58 <211 > 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 58 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Th r Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Glu Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 59 <211> 384 <212> DNA <213> Artificial Sequence <220> < 223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 59 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 c tggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaacgcc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 60 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His- Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 60 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Arg Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 61 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 61 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtcaaaac cggtagccgt ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 62 <211> 127 < 212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absnet or Histidine <400 > 62 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Ty r Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Lys Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 63 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 63 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cg gacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagcaaa ccgctgcaag cgacgttcga ggcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 64 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 64 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Lys Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 65 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 65 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcat 60 accgctgt attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga tgcgaccaag 360 caacatcacc accaccacca ctaa 384 <210> 66 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces terfezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 66 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Ty r Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Asp Ala Thr Lys Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 67 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Coding Sequence of modified protein from Mattirolomyces terfezioides with His-Tag <400> 67 atgccggatt tgagctcgtt cattactatt aagaataact ctaaccatgt ttttacgcgt 60 accgcaatct attccaaata tgccgcagtc cagtggagcc cggaaccgca gctgagcatt 120 agcccaggta aatgggacct gttcatcctg aaagacatcc tgagcatccg cggtacgtcc 180 ggctacgtgc agtaccgtgt cggcgacggt ccgggctggg tgcgtgttac ctttagcagc 240 ctggtgggtg cggacgaagt tgctgagtgg agcagcggtg atctgccgga tggctttgtt 300 ctgcaaaagc ctgtccgcac cggtagccgt ccgctgcaag cgacgttcga ggcgacccgc 360 caacatcacc accaccacca ctaa 384 <210> 68 <211> 127 < 212> PRT <213> Artificial Sequence <220> <223> Modified protein from Mattirolomyces ter fezioides with optional His-Tag <220> <221> misc_feature <222> (122)..(127) <223> Absent or Histidine <400> 68 Met Pro Asp Leu Ser Ser Phe Ile Thr Ile Lys Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Lys Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Lys Asp Ile Leu Ser Ile Arg Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Arg Val Gly Asp Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Asp Glu Val Ala Glu Trp Ser Ser Gly Asp Leu Pro 85 90 95 Asp Gly Phe Val Leu Gln Lys Pro Val Arg Thr Gly Ser Arg Pro Leu 100 105 110 Gln Ala Thr Phe Glu Ala Thr Arg Gln Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> 69 <211> 5 <212> PRT < 213> Artificial Sequence <220> <223> Amino Acid Linker <220> <221> REPEAT <222> (1)..(5) <223> May be repeated n times, where n is any nteger <400> 69 Gly Gly Gly Gly Ser 1 5 <210 > 70 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Linker <220> <221> REPEAT <222> (1)..(5) <223> May be repeated n times , where n is any integer <400> 70 Glu Ala Ala Ala Lys 1 5 <210> 71 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Consensus Sequence 1 <220> <221> misc_feature <222> (3)..(3) <223> Xaa is any amino acid <220> <221> misc_feature <222> (11)..(16) <223> Xaa is any amino acid <220> <221 > misc_feature <222> (26)..(26) <223> Xaa is any amino acid <220> <221> misc_feature <222> (33)..(34) <223> Xaa is any amino acid <220> <221> misc_feature <222> (36)..(38) <223> Xaa is any amino acid <220> <221> misc_feature <222> (41)..(43) <223> Xaa is any amino acid < 220> <221> misc_feature <222> (51)..(51) <223> Xaa is any amino acid <220> <221> misc_feature <222> (57)..(57) <223> Xaa is any amino acid <220> <221> misc_feature <222> (66)..(66) <223> Xaa is any amino acid <220> <221> misc_feature <222> (68)..(72) <223> Xaa is any amino acid <220> <221> misc_feature <222> (85)..(86) <223> Xaa is any amino acid <220> <221> misc_feature <222> (89)..(89) <223> Xaa is any amino acid <220> <221> misc_feature <222> (97)..(97) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (101)..(110) < 223> Xaa is any amino acid <220> <221> misc_feature <222> (117)..(117) <223> Xaa is any amino acid <220> <221> misc_feature <222> (120)..(120 ) <223> Xaa is any amino acid <400> 71 Met Pro Xaa Leu Ser Ser Phe Ile Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Xaa Tyr Ala Ala Val Gln Trp 20 25 30 Xaa Xaa Glu Xaa Xaa Xaa Ser Ile Xaa Xaa Xaa Lys Trp Asp Leu Phe 35 40 45 Ile Leu Xaa Asp Ile Leu Ser Ile Xaa Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Xaa Val Xaa Xaa Xaa Xaa Xaa Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Xaa Xaa Val Ala Xaa Trp Ser Ser Gly Asp Leu Pro 85 90 95 Xaa Gly Phe Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu 100 1 05 110 Gln Ala Thr Phe Xaa Ala Thr Xaa Gln 115 120 <210> 72 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence 2 <220> <221> misc_feature <222> ( 3)..(3) <223> Xaa is any amino acid <220> <221> misc_feature <222> (11)..(16) <223> Xaa is any amino acid <220> <221> misc_feature <222 > (26)..(26) <223> Xaa is any amino acid <220> <221> misc_feature <222> (33)..(33) <223> Xaa is any amino acid <220> <221> misc_feature <222> (37)..(38) <223> Xaa is any amino acid <220> <221> misc_feature <222> (41)..(41) <223> Xaa is any amino acid <220> <221 > misc_feature <222> (43)..(43) <223> Xaa is any amino acid <220> <221> misc_feature <222> (51)..(51) <223> Xaa is any amino acid <220> <221> misc_feature <222> (57)..(57) <223> Xaa is any amino acid <220> <221> misc_feature <222> (66)..(66) <223> Xaa is any amino acid < 220> <221> misc_feature <222> (68)..(70) <223> Xaa is any amino acid <220> <221> misc_feature <222> (72)..(72) <223> Xaa is any am ino acid <220> <221> misc_feature <222> (85)..(86) <223> Xaa is any amino acid <220> <221> misc_feature <222> (89)..(89) <223> Xaa is any amino acid <220> <221> misc_feature <222> (97)..(97) <223> Xaa is any amino acid <220> <221> misc_feature <222> (101)..(103) <223 > Xaa is any amino acid <220> <221> misc_feature <222> (105)..(110) <223> Xaa is any amino acid <220> <221> misc_feature <222> (117)..(117) <223> Xaa is any amino acid <220> <221> misc_feature <222> (120)..(120) <223> Xaa is any amino acid <400> 72 Met Pro Xaa Leu Ser Ser Phe Ile Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Xaa Tyr Ala Ala Val Gln Trp 20 25 30 Xaa Pro Glu Pro Xaa Xaa Ser Ile Xaa Pro Xaa Lys Trp Asp Leu Phe 35 40 45 Ile Leu Xaa Asp Ile Leu Ser Ile Xaa Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Xaa Val Xaa Xaa Xaa Pro Xaa Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Xaa Xaa Val Ala Xaa Trp Ser Ser Gly Asp Leu Pro 85 90 95 Xaa Gly Phe Val Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu 100 105 110 Gln Ala Thr Phe Xaa Ala Thr Xaa Gln 115 120 <210> 73 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Consensus Sequence 3 < 220> <221> misc_feature <222> (3)..(3) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (11)..(11) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (12)..(13) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (14 )..(14) <223> Xaa is Ser, Cys, Thr. or Met <220> <221> misc_feature <222> (15)..(15) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (16)..(16) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (26)..(26) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (33)..(33) <223> Xaa is Ser, Cys, Thr. or Met <220> <221> misc_feature <222> (37)..(37) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (38)..(38) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (41)..(41) <223> Xaa is Ser, Cys, Thr. or Met <220> <221> misc_feature <222> (43)..(43) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (51).. (51) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (57)..(57) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (66)..(66) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (68)..(68) <223> Xaa is Val, Gly, Ala , Leu.or Ile <220> <221> misc_feature <222> (69)..(69) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (70).. (70) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (72)..(72) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (85)..(86) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (89)..(89) < 223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (97)..(97) <223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222 > (101)..(101) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (102)..(102) <223> Xaa is Asp, Glu , Asn or Gln <220> <221> misc_feature <222> (103)..(103) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (105)..(105) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (106)..(106) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (107)..(107) <223> Xaa is Ser, Cys, Thr. or Met <220> <221> misc_feature <222> (108)..(108) <223> Xaa is Val, Gly, Ala, Leu.or Ile <220> <221> misc_feature <222> (108).. (108) <223> Xaa is Val, Gly, Ala, Leu, or Ile <220> <221> misc_feature <222> (109)..(109) <223> Xaa is Ser, Cys, Thr. or Met <220> <221> misc_feature <222> (110)..(110) <223> Xaa is Lys, Arg, or His <220> <221> misc_feature <222> (117)..(117) < 223> Xaa is Asp, Glu, Asn or Gln <220> <221> misc_feature <222> (120)..(120) <223> Xaa is Lys, Arg, or His <400> 73 Met Pro Xaa Leu Ser Ser Phe Ile Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Xaa Tyr Ala Ala Val Gln Trp 20 25 30 Xaa Pro Glu Pro Xaa Xaa Ser Ile Xaa Pro Xaa Lys Trp Asp Leu Phe 35 40 45 Ile Leu Xaa Asp Ile Leu Ser Ile Xaa Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Xaa Val Xaa Xaa Xaa Pro Xaa Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Xaa Xaa Val Ala Xaa Trp Ser Ser Gly Asp Leu Pro 85 90 95 Xaa Gly Phe Val Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu 100 105 110 Gln Ala Thr Phe Xaa Ala Thr Xaa Gln 115 120 <210> 74 <211> 121 <212 > PRT <213> Artificial Sequence <220> <223> Consensus Sequence 4 <220> <221> misc_feature <222> (3)..(3) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (11)..(11) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (12)..(13) < 223> Xaa is Asn or Gln <220> <221> misc_feature <222> (14)..(14) <223> Xaa is Ser or Thr <220> <221> misc_feature <222> (15)..(15 ) <223> Xaa is Asn or Gln <220> <221> misc_feature <222> (16)..(16) <223> Xaa is Leu, Val, or Ile <220> <221> misc_feature <222> (26 )..(26) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (33)..(33) <223> Xaa is Ser or Thr <220> <221> misc_feature <222> (37)..(37) <223> Xaa is Asn or Gln <220> <221> misc_feature <222> (38)..(38) <223> Xaa is Leu, Val, or Ile <220> <221 > misc_feature <222> (41)..(41) <223> Xaa is Ser or Thr <220> <221> misc_feature <222> (43)..(43) <223> Xaa is Gly or Ala <220> <221> misc_feature <222> (51)..(51) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (57)..(57) <223> Xaa is Lys or Arg < 220> <221> misc_feature <222> (66)..(66) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (68)..(68) <223 > Xaa is Gly or Ala <220> <221> misc_feature <222> (69)..(69) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (70)..(70) <223> Xaa is Gly or Ala <220> <221> misc_feature <222> (72)..(72) <223> Xaa is Gly or Ala <220> <221> misc_feature <222> (85)..( 86) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (89)..(89) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (97). .(97) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (101)..(101) <223> Xaa is Leu, Val, or Ile <220> <221> misc_feature <222 > (102)..(102) <223> Xaa is Asn or Gln <220> <221> misc_feature <222> (103)..(103) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (105)..(105) <223> Xaa is Leu, Val, or Ile <220> <221> misc_feature <222> (106)..(106) <223> Xaa is Lys or Arg <220 > <221> misc_feature <222> (107)..(107) <223> Xaa is Ser or Thr <220> <221> misc_feature <222> (108)..(108) <223> Xaa is Gly or Ala <220> <221> misc_feature <222> (109)..(109) <223> Xaa is Ser or Thr <220> <221> misc_feature <22 2> (110)..(110) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (117)..(117) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (120)..(120) <223> Xaa is Lys or Arg <400> 74 Met Pro Xaa Leu Ser Ser Phe Ile Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Xaa Tyr Ala Ala Val Gln Trp 20 25 30 Xaa Pro Glu Pro Xaa Xaa Ser Ile Xaa Pro Xaa Lys Trp Asp Leu Phe 35 40 45 Ile Leu Xaa Asp Ile Leu Ser Ile Xaa Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Xaa Val Xaa Xaa Xaa Pro Xaa Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Xaa Xaa Val Ala Xaa Trp Ser Ser Gly Asp Leu Pro 85 90 95 Xaa Gly Phe Val Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu 100 105 110 Gln Ala Thr Phe Xaa Ala Thr Xaa Gln 115 120 <210> 75 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Consensus Sequence 5 <220> < 221> misc_feature <222> (3)..(3) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (11)..(1) 1) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (26)..(26) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (51). .(51) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (57)..(57) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (66 )..(66) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (69)..(69) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (85)..(85) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (86)..(86) <223> Xaa is Asp or Glu <220> <221> misc_feature < 222> (89)..(89) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (97)..(97) <223> Xaa is Asp or Glu <220> <221> misc_feature <222> (103)..(103) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (106)..(106) <223> Xaa is Lys or Arg <220> < 221> misc_feature <222> (110)..(110) <223> Xaa is Lys or Arg <220> <221> misc_feature <222> (117)..(117) <223> Xaa is Asp or Glu <220 > <221> misc_feature <222> (120)..(120) <223> Xaa is Lys or Arg <400> 75 Met Pro Xaa Leu Ser Ser Ser Phe Il e Thr Ile Xaa Asn Asn Ser Asn His 1 5 10 15 Val Phe Thr Arg Thr Ala Ile Tyr Ser Xaa Tyr Ala Ala Val Gln Trp 20 25 30 Ser Pro Glu Pro Gln Leu Ser Ile Ser Pro Gly Lys Trp Asp Leu Phe 35 40 45 Ile Leu Xaa Asp Ile Leu Ser Ile Xaa Gly Thr Ser Gly Tyr Val Gln 50 55 60 Tyr Xaa Val Gly Xaa Gly Pro Gly Trp Val Arg Val Thr Phe Ser Ser 65 70 75 80 Leu Val Gly Ala Xaa Xaa Val Ala Xaa Trp Ser Ser Gly Asp Leu Pro 85 90 95 Xaa Gly Phe Val Leu Gln Xaa Pro Val Xaa Thr Gly Ser Xaa Pro Leu 100 105 110 Gln Ala Thr Phe Xaa Ala Thr Xaa Gln 115 120

Claims (32)

An isolated polynucleotide encoding a polypeptide having sweetness regulating activity, wherein the polynucleotide sequence has
(a) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75 a polypeptide sequence selected from the group consisting of;
(b) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , a polypeptide having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and
(c) SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID by deletion, insertion, substitution or addition of up to 24 amino acids in the group consisting of a polypeptide sequence modified from a polypeptide sequence selected from the group consisting of NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 An isolated polynucleotide encoding a selected polypeptide.
According to claim 1,
The polypeptide sequence of the polypeptide having sweet taste regulating activity is the polypeptide sequence set forth in SEQ ID NO:3, or a polypeptide sequence having at least 80% sequence identity with SEQ ID NO:3.
According to claim 1 or 2,
The polypeptide sequence comprises amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3.
According to claim 1 or 2,
An isolated polynucleotide wherein the polypeptide sequence of the polypeptide having sweet taste regulating activity comprises SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.
According to claim 4,
The polypeptide sequences of the polypeptides having the activity to regulate sweet taste are SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO : 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or an isolated polynucleotide comprising SEQ ID NO: 68.
According to claim 4 or 5,
The polynucleotide is SEQ ID NO: 23, SEQ ID NO: 25 SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49 , SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO; 65, or an isolated polynucleotide comprising SEQ ID NO: 67.
According to any one of claims 1 to 6,
An isolated polynucleotide operably linked to a heterologous regulatory element.
According to any one of claims 1 to 7,
The polynucleotide sequence further encodes a histidine tag.
An expression cassette comprising the isolated polynucleotide of any one of claims 1 to 8.
A vector comprising the isolated polynucleotide of any one of claims 1 to 8.
A host cell transformed with the vector of claim 10.
A method for producing a protein having sweetness regulating activity, comprising culturing the host cell of claim 11 in a medium under conditions for producing a protein having sweetness regulating activity.
SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID An isolated polypeptide comprising a polypeptide sequence having at least 80% sequence identity to a polypeptide sequence selected from the group consisting of NO:15, SEQ ID NO:16, and SEQ ID NO:17,
The polypeptide is optionally SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: comprises at least one substitution modification to a polypeptide sequence selected from the group consisting of 14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or
said polypeptide optionally further comprises a histidine tag;
The polypeptide is an isolated polypeptide having sweet taste regulating activity.
According to claim 13,
wherein said polypeptide comprises SEQ ID NO:3 or a polypeptide sequence having at least 80% sequence identity to SEQ ID NO:3.
According to claim 13 or 14,
said polypeptide comprising amino acid residues 1-11, 17-32, 39, 40, 45-67, 73-100 and 110-121 of SEQ ID NO:3.
According to claim 13 or 14,
The polypeptide is an isolated polypeptide comprising SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75.
17. The method of claim 16,
The polypeptide is SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or an isolated polypeptide comprising SEQ ID NO: 68.
As an isolated polynucleotide,
The polynucleotide sequence is
(a) the nucleic acid sequence set forth in SEQ ID NO:2; and
(b) a polynucleotide selected from the group consisting of a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:2;
The polynucleotide is an isolated polynucleotide that coats a polypeptide having a sweetness regulating activity.
According to claim 18,
The polypeptide sequence of the polypeptide having the sweetness regulating activity is SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO :13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 , and an isolated polynucleotide selected from the group consisting of SEQ ID NO:75.
According to claim 18 or 19,
The polynucleotide is SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67.
According to any one of claims 18 to 20,
An isolated polynucleotide operably linked to a heterologous regulatory element.
According to any one of claims 18 to 21,
The polypeptide of (a) and (b) above further comprises a nucleotide sequence encoding a histidine tag.
An expression cassette comprising the polynucleotide of any one of claims 18 to 22.
A vector comprising the polynucleotide of claim 23.
A host cell transformed with the vector of claim 24 .
A method for producing a protein having sweetness regulating activity, comprising culturing the host cell of claim 25 in a medium under conditions for producing a protein having sweetness regulating activity.
A composition comprising a combination of (a) a product for oral administration and (b) a sweetening composition comprising an isolated polypeptide,
The product for oral administration is not a Matirolomyces terfezioides truffle,
here:
(i) the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or
(ii) the polypeptide is a polypeptide of any one of claims 13-17;
The combination is a composition with enhanced sweetness compared to products for oral administration.
A method of adjusting the taste of a product for oral administration comprising combining an effective amount of a sweetening composition comprising an isolated polypeptide with the product for oral administration, comprising:
here:
(i) the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or
(ii) the polypeptide is a polypeptide of any one of claims 13 to 17;
The product for oral administration is not Matyrolomyces terpegioides truffle,
Wherein the combination has enhanced sweetness compared to products for oral administration.
The method of claim 27 or 28,
The composition or method of claim 1, wherein the product for oral administration is a food, beverage, dietary supplement composition, or pharmaceutical composition.
The method of claim 27 or 28,
The product for oral administration may be used in confectionery; sweet bakery products, ready-made sweet bakery mixes for preparing sweet bakery products; pie fillings and other sweet fillings, gelatins and puddings; frozen desserts; yogurt; snack bar; bread products; pre-made bread mixes for preparing bread products; sauces, syrups and dressings; sweet spread; confectionery products; and a food selected from the group consisting of sweetened breakfast cereals.
The method of claim 27 or 28,
Products for oral administration include carbonated beverages; non-carbonated beverages; and a beverage concentrate.
(a) obtaining a composition comprising a polypeptide having sweet taste regulating activity;
(b) a method for purifying a polypeptide having sweetness modulating activity comprising purifying the composition via hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC),
here:
(i) the polypeptide is SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID comprises an amino acid sequence having at least 80% sequence identity to a polypeptide selected from the group consisting of NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; or
(ii) the polypeptide is a polypeptide of any one of claims 13-17.

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